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Regulation of GTPase and adenylate cyclase activity by amyloid β-peptide and its fragments in rat brain tissue
Brain Research 850 Ž1999. 179–188 www.elsevier.comrlocaterbres
Research report
Regulation of GTPase and adenylate cyclase activity by amyloid b-peptide and its fragments in rat brain tissue Ursel Soomets a,b , Riina Mahlapuu b , Roya Tehranian a , Juri ¨ Jarvet c , Ello Karelson b , b a a,d c ¨ Mihkel Zilmer , Kerstin Iverfeldt , Matjaz Zorko , Astrid Graslund , Ulo Langel a, ) ¨ a
Department of Neurochemistry and Neurotoxicology, Arrhenius Laboratories, Stockholm UniÕersity, S-106 91 Stockholm, Sweden b Department of Biochemistry, Tartu UniÕersity, EE2400 Tartu, Estonia c Department of Biophysics, Stockholm UniÕersity, S-106 91 Stockholm, Sweden d Institute of Biochemistry, Medical Faculty, UniÕersity of Ljubljana, VrazoÕ trg 2, 1000 Ljubljana, SloÕenia Accepted 21 September 1999
Abstract Modulation of GTPase and adenylate cyclase ŽATP pyrophosphate-lyase, EC 4.6.1.1. activity by Alzheimer’s disease related amyloid b-peptide, AbŽ1–42., and its shorter fragments, AbŽ12–28., AbŽ25–35., were studied in isolated membranes from rat ventral hippocampus and frontal cortex. In both tissues, the activity of GTPase and adenylate cyclase was upregulated by AbŽ25–35., whereas AbŽ12–28. did not have any significant effect on the GTPase activity and only weakly influenced adenylate cyclase. AbŽ1–42., similar to AbŽ25–35., stimulated the GTPase activity in both tissues and adenylate cyclase activity in ventral hippocampal membranes. Surprisingly, AbŽ1–42. did not have a significant effect on adenylate cyclase activity in the cortical membranes. At high concentrations of AbŽ25–35. and AbŽ1–42., decreased or no activation of adenylate cyclase was observed. The activation of GTPase at high concentrations of AbŽ25–35. was pertussis toxin sensitive, suggesting that this effect is mediated by GirGo proteins. Addition of glutathione and N-acetyl-L-cysteine, two well-known antioxidants, at 1.5 and 0.5 mM, respectively, decreased AbŽ25–35. stimulated adenylate cyclase activity in both tissues. Lys-AbŽ16–20., a hexapeptide shown previously to bind to the same sequence in Ab-peptide, and prevent fibril formation, decreased stimulation of adenylate cyclase activity by AbŽ25–35., however, NMR diffusion measurements with the two peptides showed that this effect was not due to interactions between the two and that AbŽ25–35. was active in a monomeric form. Our data strongly suggest that Ab and its fragments may affect G-protein coupled signal transduction systems, although the mechanism of this interaction is not fully understood. q 1999 Elsevier Science B.V. All rights reserved. Keywords: GTPase; Adenylate cyclase; Amyloid b-peptide; Alzheimer’s disease
1. Introduction In several recent reports, the alteration of the cellular signal transduction system in the brain of patients with the Alzheimer’s disease ŽAD. has been pointed out. It has been shown w11,36,37x that the overall activity of G-proteins is altered in the post-mortem brain of AD patients. Impaired coupling of adenosine A1 receptors w48x, badrenergic receptors w19x and other receptors to G-proteins in different tissues such as brain and skin of AD patients has been reported. The reasons for such alterations in cellular signal transduction in brain of AD patients are not )
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clarified yet, although the change in parameters of oxidative stress has been suggested as one of the possible causes w23x. Various fragments of the amyloid precursor protein, APP, affect important functions in the brain. The part of the intracellular C-terminus of APP Žsequence 657–676. has been shown to interact with brain Go-proteins w34x. It has been suggested that abnormal APP-Go signalling can be involved in the AD process and that APP has a potential receptor function w35x. Recently, Brouillet et al. w4x showed the regulation of Go GTPase activity by APP in a caveolae-like compartments specialised in signal transduction. Also, Smine et al. w41x have demonstrated that presenilin-1, a product of the familial AD gene, interacts with brain Go proteins. The described specific interactions of
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APP with presenilins w50x, may play a significant role in intramembraneous proteolysis of APP as well as of abnormal signal transduction in early onset AD ŽFAD.. A 39–43 amino acids long proteolytic fragment of APP, amyloid b-peptide ŽAb ., is a major component of the senile plaques associated with AD. The sequences of AbŽ1–42. and the peptides derived from it used in the present study are shown in Fig. 1. The most toxic fragments of Ab, AbŽ25–35. and AbŽ12–28., modulate neuronal function and immune and inflammatory responses in several cell types w36,37x. Although these peptides have not been found as naturally occurring degradation products of APP, they are widely used in studies of the mechanisms of action of Ab in in vitro studies. AbŽ1–40. and AbŽ25– 35. peptides both disrupt carbachol-induced M1 muscarinic cholinergic signal transduction in cortical neurons w22x suggesting that Ab peptides interfere with muscarinic receptor coupling to G-proteins. These results indicate that Ab plays an important role in the impairment of cholinergic transmission that occurs in AD, probably with the involvement of free radicals in the mechanism w22,38x. It has been suggested that the toxicity of Ab and its fragments is well-correlated with the degree of aggregation of these peptides. The aggregation of AbŽ1–40. and Ž1–42. peptides into fibrils has been shown to depend on pH, concentration, and the incubation time in aqueous solutions in vitro w6,16x. The polymerisation of full-length Ab into b-amyloid fibrils could be inhibited by the pentapeptide AbŽ16–20. with the sequence KLVFF as well as with its better water soluble analogue Lys-AbŽ16–20., KKLVFF w45–47x. The KLVFF-sequence is the region in Ab that most efficiently binds to Ab, and is necessary for fibril formation w45x. The mechanism of interaction of the Ab peptide with possible different signal transduction routes is, however, not clear today. Due to the cationicr amphiphilic nature of Ab-peptides, which may allow them to interact with and penetrate biological membranes w12,30x, it is not excluded that their action is mediated via G-proteins, i.e., via a receptor-independent mode. In the neurons the mechanism of neurotoxicity of Ab fragments appears also to involve generation of free radicals, induction of reactive oxygen species and to cause
lipid peroxidation w3,7,8,51,52x. Different reports have shown cellular release of peroxides, superoxide and nitric oxide in response to Ab and its fragments treatment w3,20x. Spontaneous generation of free radicals by AbŽ1–40. and AbŽ25–35. themselves in free-cell system has been reported w17x. In contrast, the recent study by Dikalov et al. w10x showed that those peptides do not yield radical adducts in the same system. It is shown that AbŽ1–40. and AbŽ25–35. can have similar or different cytotoxic mechanisms in different cell types w31x. Interactions of stimulated 7TM receptors with the asubunits of G-proteins result in alteration of GTPase activity leading to different cellular events, such as the synthesis of second messengers, e.g., cyclic AMP, generated by a membranous enzyme adenylate cyclase. It has been shown that several short amphiphilic peptides are able to directly affect the functions of G-proteins w2,33,40,53x in this way mimicking the action of native membrane proteins. In the present study, we have examined the influence of the APP-derived peptides, AbŽ25–35., AbŽ12–28., and AbŽ1–42. on the activity of GTPase and adenylate cyclase in membranes from rat ventral hippocampus and cerebral cortex. The influence of two well-known antioxidants on the enzymatic activity of adenylate cyclase was characterised as well. Since the Ab-related toxicity is associated with an aggregated state of the peptide, we studied the possible aggregation of the fragments AbŽ25–35. and AbŽ12–28. by diffusion measurements using NMR. The possible interaction of Lys-AbŽ16–20., a motif in Ab peptide essential for Ab–Ab fibril formation and binding w45,46x, with AbŽ25–35. was studied by the same method, since we show that the modulation of GTPase and adenylate cyclase activity by AbŽ25–35. was altered by LysAbŽ16–20.. As control, the effect of scrambled AbŽ25–35. on the activity of GTPase and adenylate cyclase was tested. 2. Materials and methods 2.1. Materials Amino acid derivatives were purchased from Nova Chemical, UK. Tritiated cyclic AMP Ž59 Cirmmol. was obtained from Amersham, UK. w g-32 PxGTP Ž5000 Cirmmol. was from NEN research products, UK. Na 2 ATP, GTP, cyclic AMP, phosphoenol pyruvate, bacitracin, bovine serum albumin, glutathione, N-acetyl-L-cysteine and pertussis toxin ŽPTX. were from Sigma, USA, or Fluka, Switzerland. All reagents used were of highest analytical grade. AbŽ1–42. was purchased from Bachem, Switzerland. 2.2. Peptide synthesis
Fig. 1. Amino acid sequences of AbŽ1–42., AbŽ12–28., Lys-AbŽ16–20., AbŽ25–35., and AbŽ25–35.-scrambled peptides.
All the shorter synthetic fragments of Ab peptides were synthesized in a stepwise manner in a 0.1-mmol scale on
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an Applied Biosystem Model 431A peptide synthesiser on a solid support using N, N X-dicyclohexylcarbodiimidehydroxybenzotriazole activation strategy. tert-Butyloxycarbonyl amino acids were coupled as hydroxybenzotriazole esters to a phenylacetamidomethyl-resin Ž0.6 mmol amino groups per gram resin, Novabiochem, Switzerland. to achieve the C-terminal free carboxylic acids. The peptides were finally cleaved from the resin with liquid HF at 08C for 30 min. Deprotection of the side chains, cleavage of the peptides and purification on HPLC have been described in detail earlier w24x. The purity of the peptides was ) 99% as demonstrated by HPLC on an analytical Nucleosil 120-3 C 18 reversed-phase column Ž0.4 cm = 10 cm.. The molecular masses of the peptides were determined by a plasma desorption mass spectrometry ŽBioion 20, Applied Biosystems. and the calculated values were obtained in each case. 2.3. Preparation of peptide solutions Lyophilised peptides were freshly resuspended in the assay buffer at room temperature, diluted and assayed immediately for adenylate cyclase and GTPase activity measurements. 2.4. GTPase assay Measurement of GTPase activity was performed radio metrically according to Cassel and Selinger w9x, with the modifications suggested by McKenzie w32x. Membranes from rat brain frontal cortex and hippocampus were obtained according to the protocol of McKenzie w32x with minor modifications as described previously w53x. To the diluted membranes Žfinal protein concentration in the assay mixture was 0.2 mgrml, as determined by the method of Lowry et al. w26x. the ice-cold reaction cocktail containing ATP Ž1 mM., 5X-adenylylimido-diphosphate Ž1 mM., ouabain Ž1 mM., phosphocreatine Ž10 mM., creatine phosphokinase Ž2.5 Unitsrml., dithiothreitol Ž4 mM., MgCl 2 Ž5 mM., NaCl Ž100 mM., and trace amounts of w g-32 PxGTP to give 50.000–100.000 c.p.m. in an aliquot of the reaction cocktail Žwith the addition of cold GTP to give the required total concentration of GTP of 0.5 mM. was added. Incubation medium was standard TE-buffer Ž10 mM Tris– HCl q 0.1 mM EDTA., pH 7.5. Background low-affinity hydrolysis of w g-32 PxGTP was assessed by incubating parallel tubes in the presence of 100 mM GTP. Blank values were determined by the replacement of the membrane solution with assay buffer. GTPase reaction was started by transferral of the reaction mixtures to 308C water bath for 12 min. Unreacted GTP was removed by the 5% suspension of the activated charcoal in 20 mM H 3 PO4 . The radioactivity of the released radioactive phosphate was determined in the LKB 1214 Rackbeta or in Packard 3255
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liquid scintillation counter. The basal GTPase activity of the rat brain cortical membrane preparation was 65 " 3 pmolrminrmg protein and that of the ventral hippocampal membranes was 52 " 4 pmolrminrmg protein. 2.5. Adenylate cyclase assay Membranes of ventral hippocampus and frontal cortex were prepared from Wistar rats Ž200–300 g., according to previously published procedures w49x. Homogenates Žin 8 mM HEPES-Na, pH 7.4. of precooled ventral hippocampus were diluted, stirred on ice for 30 min and centrifuged for 6 min at 1600 = g. The pellets were resuspended in ice-cold protein-buffer Ž4 mM HEPES-Na, 1.5 mM theophylline, 8.25 mM MgCl 2 , 0.75 mM EGTA, 7.5 mM KCl, 100 mM NaCl, pH 7.4. to a final protein concentration of 0.6–0.8 mgrml. The basal adenylate cyclase activity was assayed at 0.04 mgrml of membrane protein in reactionbuffer, additionally containing Žin protein buffer. 100 mgrml bacitracin, 0.03% bovine serum albumin, 10 mM phosphoenol-pyruvate and 30 mgrml pyruvate kinase w49x. In all experiments, the peptides, dissolved in the reaction buffer, were added to the assay mixture 2 min before the reaction was initiated by 10 mM ATPr10 mM GTP. The reaction at 308C was terminated after 15 min by the addition of 100 mM EDTA, followed by boiling the samples for 3 min. The cyclic AMP content in the tubes was measured by a competitive protein saturation assay using cyclic AMP-binding protein from bovine adrenal cortex w5x. The basal levels of the adenylate cyclase activity in ventral hippocampus and in frontal cortex were 52 " 3 and 76 " 1 pmol cyclic AMPrminrmg protein, respectively. The protein content of the membrane preparations was determined according to Lowry et al. w26x. 2.6. Incubation of tissue membranes with antioxidants We have examined the effects of the antioxidants, glutathione and N-acetyl-L-cysteine, on the basal activity of adenylate cyclase as well antioxidant induced alterations in the modulation of adenylate cyclase activity by AbŽ25– 35.. The effect was measured as a difference in the amount of cAMP, produced by membranous adenylate cyclase in the presence or absence of 10y7 M AbŽ25–35. and in the conditions where glutathione Žfinal concentration 1.5 mM. or N-acetyl-L-cysteine Ž0.5 mM. were added to the medium before the peptide, and shown as percentage of changes in cAMP production against unaffected Žbasal. activity Žs 100%.. 2.7. PTX catalysed ADP-ribosylation PTX was activated by treatment with 50 mM dithiotreitol ŽDTT. for 30 min at 378C. For the ADP-ribosylation reaction, the membranes from brain cortex and ventral
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hippocampus were treated with 10 mgrml of PTX in the 10 mM HEPES-Na or TE buffer ŽpH 7.4., containing 3 mM NADq, 20 mM thymidine, 1 mM ATP and 100 mM GTP w27x. The treatment was initiated by transferring the tissue membranes to a 378C water bath for 30 min. After the incubation, the membranes were diluted two-fold and centrifuged at 10 000 = g for 45 min. The obtained pellet was resuspended with ice-cold buffer and used for the GTPase or adenylate cyclase assay experiments.
Table 1 Translational diffusion coefficients of Ab fragments Lys-AbŽ16–20. and AbŽ25–35., determined using pulsed field gradient NMR spectroscopy Experimental values of the translational diffusion coefficient D along with calculated molecular weights are listed with theoretical molecular weights. Samples used were 300 mM peptide in 70 mM phosphate buffer, pH 7.0, at 258C.
Lys-AbŽ16–20. AbŽ25–35.
10 6 Drcm2 sy1
Molecular weight rDa
Molecular weight Žtheor.rDa
3.13"0.05 2.76"0.05
760"50 1230"100
723.9 1063.3
2.8. Measurements of translational diffusion coefficients by NMR NMR spectra were acquired on a Varian Inova 600 MHz spectrometer at 298 K. Peptide samples were prepared in 70 mM phosphate buffer, pH 7.0, with concentrations ranging from 300 mM to 1 mM. The translational diffusion coefficient was measured using pulsed field gradient longitudinal eddy-current delay type pulse sequence w13,43x. The gradient strength was calibrated using a HDO signal in 99.9% D 2 O and literature value for diffusion coefficient of 1.9 = 10y5 cm2 sy1 w25x. In the diffusion studies, the integral of the methyl resonances between 0.97 and 0.71 ppm was used for the Lys-AbŽ16–20. peptide and the integral of the Met methyl signal at 2.10 ppm was used for the AbŽ25–35. fragment. In diluted samples the diffusion coefficient D is related to the molecular hydrodynamic radius R h according to the Stokes-Einstein law: D s k B Tr6ph R h , where k B is the Boltzmann constant, T is the absolute temperature, and h is the viscosity of water. From the known specific volume of proteins, V s 0.73 cm3 gy1 and assuming a hydration ˚ one can calculate the molecular weight layer rw s 1.8 A, of a peptide Mw from the experimentally determined hydrodynamic radius R h : Mw s Ž4r3. p Ž R h y rw . 3 NAVy1. Translational diffusion coefficients of Ab fragments LysAbŽ16–20. and AbŽ25–35. are shown in Table 1.
3. Results
3.1. GTPase actiÕity The effects of Ab peptides on the activity of GTPase in membranes isolated from rat ventral hippocampus and frontal cortex are illustrated in Fig. 2. In membranes from both brain regions, the GTPase activity was stimulated by AbŽ25–35. peptide and increased by 95% and 104%, respectively, at 100 mM concentration ŽFig. 2A and B.. The EC 50 value of the activation was 29 " 4 mM in the ventral hippocampal membranes Ž n H s 1.7 " 0.3. and 26
" 3 mM Ž n H s 2.4 " 0.7. in the cortical membranes. In contrast, AbŽ25–35. peptide with the scrambled sequence ŽFig. 1. used as control at the all tested concentrations Ž10y9 to 10y5 M. had no effect on GTPase activity Ždata not shown.. The effects of AbŽ1–42. on GTPase activity from both brain regions were also investigated and are shown in Fig. 2. The EC 50 value of the stimulatory effect of AbŽ1–42. was 1.36 " 0.25 mM in the ventral hippocampal membranes and 1.33 " 0.23 mM in the cortical membranes. The concentration-dependent effect of AbŽ1– 42. on GTPase activity was bell-shaped and declined at higher concentrations of the peptide. AbŽ12–28. had no effect on GTPase activity in the concentrations tested. The hexapeptide, Lys-AbŽ16–20., which had no effect on its own, slightly decreased the maximal effect of AbŽ25–35. to 60% and 75% in the ventral hippocampal and cortical membranes, respectively, leaving the values of EC 50 and n H practically unchanged. PTX catalysed ribosylation of the membranes totally prevented activation of GTPase by AbŽ25–35. peptide ŽFig. 2A and B., suggesting that AbŽ25–35. activates GirGo but not Gs type of G-proteins.
3.2. Adenylate cyclase actiÕity In Fig. 3A and B, the effects of AbŽ25–35., AbŽ1–42., AbŽ12–28. and Lys-AbŽ16–20. on the activity of adenylate cyclase in the membranes from rat ventral hippocampus and frontal cortex are presented. In the membranes from both brain regions, AbŽ25–35. exhibited a bellshaped dose-dependence effect on the activity of adenylate cyclase. In ventral hippocampus ŽFig. 3A., AbŽ25–35. stimulated the enzyme at the lower concentrations with the maximal value of 33 " 2% at 0.1 mM ŽEC 50 s 1.7 nM.. At higher concentrations of AbŽ25–35. Žup to 10 mM., no stimulatory effect of the peptide could be observed. In the frontal cortical membranes ŽFig. 3B., the dose–response curve for AbŽ25–35. was similar ŽEC 50 s 2.7 nM. to that in the ventral hippocampus, but the maximal activation was lower Ž23 " 7% at 0.1 mM.. The activity of adenylate cyclase was not affected by scrambled AbŽ25–35. peptide
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Fig. 2. Effects of AbŽ25–35. Žv ., AbŽ1–42., ŽX., AbŽ12–28. Ž'., and Lys-AbŽ16–20. Ž^., on GTPase activity in rat ventral hippocampal ŽA. and frontal cortical ŽB. membranes: the effect of 100 mM Lys-AbŽ16–20. ŽI., and PTX catalysed ADP-ribosylation Žmembranes were treated at 378C with 10 mgrml PTX for 30 min. Ž`., on the activation of GTPase by AbŽ25–35. is also shown. The data represent the average of four independent experiments. S.E.M. never exceeded 10%; error bars are not shown for clarity. Curves for the activation of GTPase by AbŽ25–35. in the absence and in the presence of Lys-AbŽ16–20. were obtained by non-linear regression fitting of the single phase dose–response equation using Prism computer program ŽGraphPad, USA..
tested in the same concentrations as AbŽ25–35. Ždata not shown.. The effect of AbŽ1–42. on adenylate cyclase activity was also studied in ventral hippocampus and was similar to the effects of AbŽ25–35.. AbŽ1–42. stimulated the enzyme with the maximum value of 45 " 4% at 0.05 mM ŽEC 50 s 2 nM.. Further increase in the concentration of the peptide reversed the stimulatory effect on the enzymatic activity. In the frontal cortical membranes, AbŽ1–42. had no effect on the activity of adenylate cyclase in the concentrations tested. AbŽ12–28. slightly increased the activity of adenylate cyclase in ventral hippocampus, whereas, in the frontal cortex the effects of this peptide were not statistically significant. The hexapeptide Lys-
AbŽ16–20. had no effect on adenylate cyclase activity in ventral hippocampus and cortex. Since the hexapeptide Lys-AbŽ16–20. could decrease the stimulatory effect of AbŽ25–35. on GTPase activity ŽFig. 2., we studied whether the hexapeptide could interfere with AbŽ25–35. induced stimulation of adenylate cyclase activity. Membranes from ventral hippocampus were incubated with 0.1 mM AbŽ25–35. or AbŽ12–28. for 2 min before Lys-AbŽ16–20. Ž1 nM–10 mM. was added. These experiments demonstrate ŽFig. 5A and B. that the stimulatory effects of AbŽ25–35. on the adenylate cyclase activity in the membranes from ventral hippocampus and cortex were completely inhibited by Lys-AbŽ16– 20. at higher concentrations. This hexapeptide also re-
Fig. 3. Effects of AbŽ25–35. Žv ., AbŽ1–42. ŽX., AbŽ12–28. Ž'., and Lys-AbŽ16–20. Ž`., on adenylate cyclase activity in rat ventral hippocampal ŽA. and frontal cortical ŽB. membranes. The data represent mean " S.E.M. of three independent experiments Ž100% corresponds to the basal adenylate activity..
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Fig. 4. The effect of AbŽ25–35. on adenylate cyclase activity in the rat ventral hippocampal ŽA. and frontal cortical ŽB. membranes treated at 378C with 10 mgrml PTX for 30 min before the experiment. The data represent mean " S.E.M. of three independent experiments Ž100% corresponds to the basal adenylate activity..
versed the stimulation of adenylate cyclase activity by AbŽ12–28. in the ventral hippocampal membranes Ždata not shown.. 3.2.1. Effect of glutathione and N-acetyl-L-cysteine on adenylate cyclase actiÕity Reduced glutathione ŽGSH. has previously been shown to protect SK-N-SH human neuroblastoma cells from AbŽ25–35. toxicity w15x. We examined the effect of glutathione and N-acetyl-L-cysteine on the activity of adenylate cyclase in rat ventral hippocampal and cerebral cortical membranes. Glutathione at 1.5 mM alone inhibited the basal activity of adenylate cyclase Ž100%. in ventral hippocampal membranes by 32.8 " 5.2% and in cortical membranes by 7.4 " 3.0%. Surprisingly, N-acetyl-L-cysteine increased the adenylate cyclase basal activity in ventral hippocampus and in frontal cortex by 19.4 " 3.5% and 16.3 " 3.6%, respectively. Incubation of membranes with antioxidants and 0.1 mM AbŽ25–35. abolished or decreased the stimulation of
adenylate cyclase activity by this fragment of Ab. Glutathione at 1.5 mM, together with 0.1 mM AbŽ25–35., showed the decrease of basal enzyme activity by 22.5 " 8.3% in ventral hippocampal and 9.7 " 0.9% in cortical membranes. N-acetyl-L-cysteine at 0.5 mM decreased the AbŽ25–35. stimulated adenylate cyclase activity from 32.5 " 2.1% to 8.2 " 1.4% in ventral hippocampus and in frontal cortex from 22.6 " 7.0% to 6.0 " 1.0% over basal activity. 3.3. Effect of PTX on actiÕity of adenylate cyclase To further elucidate the mechanismŽs. behind the bellshaped dependency of the adenylate cyclase activity on the AbŽ25–35., we investigated if the effect was PTX-sensitive on membranes prepared from hippocampus and cortex. Fig. 4 shows that in PTX-treated membranes a dosedependent stimulatory effect of AbŽ25–35. was retained both in ventral hippocampus Žmaximal effect 30.7%, EC 50s s 11.1 nM. and cortex Žmaximal effect 33.2%, EC 50 s 2.55 nM., whereas no attenuation of the stimula-
Fig. 5. The effect of Lys-AbŽ16–20., on the stimulated adenylate cyclase activity. ŽA. Rat ventral hippocampal membranes stimulated by 10y7 M of AbŽ25–35.. ŽB. Rat frontal cortical membranes stimulated by 10y8 M of AbŽ25–35.. The data represent mean " S.E.M. of three independent experiments Ž100% corresponds to the basal adenylate activity..
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tion at higher concentrations Žup to 10 mM. of AbŽ25–35. could be observed. 3.4. NMR measurements The propensity of the peptides to aggregate in aqueous solution was investigated by diffusion studies using NMR. The experimentally determined translational diffusion coefficients and calculated molecular weights along with the theoretical molecular weights for Lys-AbŽ16–20. and AbŽ25–35. peptides are summarised in Table 1. The results show that both peptides have values close to that expected for a monomeric state under the conditions used, i.e., the same pH and somewhat higher concentrations than those used in the activity studies. For AbŽ12–28., under the same conditions, aggregation was immediate and to such extent that NMR-based diffusion measurements became impossible due to the large line width in the NMR spectra Ždata not shown.. Measurements were performed also at pH 5.0 for all three peptides with similar results as at pH 7.0. In the 1:1 mixture of Lys-AbŽ16–20. and AbŽ25–35., the diffusion coefficients of the two peptides were determined as 3.13 = 10y6 and 2.76 = 10y6 cm2 sy1 , respectively, i.e., unchanged from when they were measured in separate samples ŽTable 1.. The results indicate that two peptides do not interact with each other. 4. Discussion Cerebral deposition of amyloid, an insoluble extracellular precipitate mainly consisting of Ab peptide, seems to be a central event in AD. The mechanisms by which the amyloid deposition is associated with dementia in AD is rather elusive. Alterations in signal transduction mechanisms involving different G-protein coupled receptors have been shown in AD. Here, we discuss the effects of AbŽ1– 42. and its shorter fragments, AbŽ25–35. and AbŽ12–28., on the activity of two key enzymes, GTPase and adenylate cyclase, in cellular signal transduction. In membrane preparations from hippocampus and cortex, AbŽ25–35. and AbŽ1–42. stimulated the activity of membrane-bound GTPase. The maximal effect of AbŽ25– 35., observed at 100 mM concentrations, was two-fold above the basal activity of the enzyme. The maximal effect of AbŽ1–42. on GTPase activity was about 1.5-fold above basal activity and detected at much lower concentration Ž5–7 mM. of the peptide. The effect of AbŽ1–42. on the GTPase activity was bell-shaped. AbŽ1–42. stimulation of GTPase activity declined at concentrations above 5 mM both in hippocampal and cortical membranes. AbŽ1–42. activation of adenylate cyclase in hippocampal membranes was similar to that of AbŽ25–35.. AbŽ1–42. stimulated adenylate cyclase to 30%–40% above basal activity in nanomolar concentration range, however, above 100 nM concentration, the effect was attenuated. In membranes from the cerebral cortex, the
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full-length Ab peptide had no significant effects on adenylate cyclase activity. There might be several explanations for the distinct effects of AbŽ25–35. and the full-length AbŽ1–42. on the activity of adenylate cyclase and GTPase. As it has been proposed the AbŽ25–35. and the full-length peptide may activate different signalling systems and therefore have different effects on the systems used in our experiments. Another explanation for the different effects might be due to the different heterogeneity of G-proteins between these brain regions. A third explanation may lie in the fact that the two peptides may interact differently with membrane components, for example, Mason et al. w30x, showed recently that AbŽ1–42. and AbŽ1– 40. behaved differently within the membrane. AbŽ1–42. interacted only with the membrane lipid bilayer hydrocarbon core, both in aggregated and soluble form, whereas AbŽ1–40., in its aggregated form interacts with hydrated surface of the membrane and is associated with the hydrocarbon core when it is soluble. Although the stimulation of GTPase by AbŽ25–35. was similar in both tissues, the value of Hill coefficient Ž n H . for this activatory peptide was somewhat higher in the frontal cortex than in the ventral hippocampus, 2.4 and 1.7, respectively. The different heterogeneity in G-protein subunits in these tissues, the multiple binding to G-proteins or interaction between the peptide and differently composed membranes could provide an explanation for these different n H values. It has been shown previously that the accumulation, conformation and orientation of Ab peptides in cell membranes can be affected by a heterogeneous composition of membrane lipids, proteins and carbohydrates w44x. A dual response of adenylate cyclase to the neurotoxic AbŽ25–35. in membranes of rat ventral hippocampus and frontal cortex was found. This response includes a consistent, 20%–30% enhancement of adenylate cyclase activity at low concentrations and less or no effects at higher concentrations of the peptide. Such a biphasic mechanism of action at adenylate cyclase has been previously demonstrated for several neuronal hormones such as NPYŽ17–36., glucagon and thrombin w14,28,39x. Both the GirGo-proteins are present in large quantities in the mammalian brain and serve as the major contributors to the high affinity GTPase activity w1,36x. Since the significant increase of GTPase activity and the reduced stimulation of adenylate cyclase activity were detected at relatively high concentrations Ž10–100 mM. of AbŽ25–35., one could assume that the peptide inhibits adenylate cyclase at higher concentrations via activation of inhibitory GirGo-proteins. In fact, in PTX-treated membranes, AbŽ25–35. stimulation at higher concentrations was no longer attenuated ŽFig. 4.. We suggest that the inhibitory phase in the pronounced bell-shaped effect of AbŽ25–35. on the brain adenylate cyclase activity Žcf. Fig. 3A and B. may be directly mediated by low-affinity isoformŽs. of Gi-proteins. This
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suggestion is strongly corroborated by the ability of PTX to totally prevent the activation of GTPase by AbŽ25–35. both in the membranes from the frontal cortex and from the ventral hippocampus ŽFig. 2. and, additionally, by the fact that AbŽ25–35. shares some structural and amphipathic features of mastoparan w2x, a known direct activator of GirGo a-subunit w18x. One possibility would be that potent stimulation of GTPase activity by high concentrations of AbŽ25–35. is specifically associated with those Gi-isoforms that inhibit the adenylate cyclase activity in the ventral hippocampus and frontal cortex. The mechanism of the activation of adenylate cyclase at low concentrations of AbŽ25–35. thus remains to be clarified, however, our results suggest that the activation of adenylate cyclase by AbŽ25–35. is not mediated via Gs-proteins. On the other hand, the suppression of cyclic AMP-signalling pathway at higher concentrations of the peptide by the stimulation of inhibitory GirGo-proteins seems to be well-supported by our study. An interesting observation in this study is that a decrease in the AbŽ25–35. stimulated adenylate cyclase activity occurs in the presence of glutathione and N-acetylL-cysteine. The significant cutback of this activation by the free radical scavengers may show that a redox component is involved in the mechanism of stimulation of adenylate cyclase activity by AbŽ25–35.. On the other hand, AbŽ25–35. has been shown to increase intracellular levels of reactive oxygen species only at the higher concentration than used by us. Glutathione decreased and N-acetyl-L-cysteine increased basal activity of adenylate cyclase in both rat brain tissues, i.e., acting differentially: the reasons for this are not clear yet. The scavengers may influence on enzyme activity by different mechanism, glutathione, for example, through the binding to the glutamate receptors. The NMR experiments show that the effects of AbŽ25– 35. are due to monomeric form of the peptide. In contrast, no effects on regulation of GTPase and adenylate cyclase activity were found for the aggregated AbŽ12–28.. These results support the hypothesis that the regulatory effects studied here are not directly related to the general toxicity of the Ab peptide and its fragments in aggregated forms, but may be due to more specific interactions, like direct interactions with GirGo proteins, as discussed above, or interaction with other components of the signal transduction systems, yet unknown. The peptide AbŽ12–28. and the hexapeptide LysAbŽ16–20. did not show any detectable effect on the GTPase activity in the tested brain regions under the specified reaction conditions. However, the hexapeptide was capable of slightly reducing the maximal activatory effect of AbŽ25–35. on the GTPase activity in the membranes from both tissues. These results suggest that the hexapeptide could directly or indirectly interfere with the interaction of AbŽ25–35. with a-subunitŽs. of G-proteins in the membranes. Our NMR-studies demonstrate the absence of the direct interaction between Lys-AbŽ16–20.
and AbŽ25–35. in a water environment, supporting the idea of indirect and probably non-specific interference of the hexapeptide. Additionally, short peptidic ligands, capable of binding efficiently to the neurotoxic AbŽ25–35., have not been identified yet although some penta- to decapeptides have been shown previously to interact with other Ab fragments as well as with full-length AbŽ1–40. w42x. The fragment AbŽ12–28. and the hexapeptide LysAbŽ16–20. generate only a slight modification of adenylate cyclase activity. Addition of the hexapeptide to the membranes after their incubation with 0.01 mM AbŽ25– 35., leads to the concentration-dependent inhibition of the stimulatory phase in the bell-shaped regulatory effect of AbŽ25–35. on adenylate cyclase activity in both brain regions ŽFig. 5.. When the hexapeptide was added to the reaction media before the AbŽ25–35., the stimulation of adenylate cyclase activity by AbŽ25–35. in both regions was suppressed in a similar way Ždata not shown.. The inhibitory phase in the adenylate cyclase modulation by higher Ž10y5 M. concentrations of AbŽ25–35. was less affected by the hexapeptide ligand. This is in accordance with a very small effect of Lys-AbŽ16–20. on the activation of GTPase by AbŽ25–35. and also corroborates the suggestion that the inhibitory effect of higher concentration of AbŽ25–35. on adenylate cyclase activation is exerted through GirGo-proteins. The attenuation of the stimulatory phase in the biphasic effect of AbŽ25–35. on the adenylate cyclase activity in the brain membranes in the presence of the hexapeptide might be due to the interaction of the cationic hexapeptide with Ga-proteins via a receptor-independent mode. Another explanation could be the action of partly hyper-aggregated intermediates of the AbŽ25–35. disrupting the cyclic AMP synthesis and leading to dysfunction of signal transduction as observed in post-mortem AD brain w21x rather than a simultaneous and additive inhibition of adenylate cyclase by the hexapeptide and AbŽ25–35.. In summary, our data show a response of G-protein, probably GirGo , coupled cyclic AMP-signalling system to the neurotoxic AbŽ25–35. in the membranes from the studied brain regions. The mechanism of action of AbŽ25– 35. may involve different subtypes of regulatory G-proteins but also other signal transduction mechanisms. The ability of Ab and its neurotoxic fragments to initiate membrane lipid peroxidation and to enhance oxidative stress in primary neuronal cultures has recently been reported w29x. Our present data suggest, in addition to the widely proposed role of amyloid aggregation, other mechanisms of amyloid peptide interaction, such as the influence on signal transduction, in Alzheimer’s disease. Acknowledgements This work was supported by Swedish Institute Žstipend to U.S.., by Wenner-Gren Center, Stockholm, Sweden
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Žstipend to M.Z.., by Structural Joint European Project S-JEP-09270-95, and by grants from Swedish Research Council for Natural Sciences ŽNFR., from Royal Swedish Academy, and from Ministry of Science and Technology of Republic of Slovenia.
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References w1x T. Asano, H. Shinohara, R. Morshita, K. Kato, Immunochemical and immunohistochemical localization of the G-protein Gi1 in rat central nervous tissues, J. Biochem. 108 Ž1990. 988–994. ¨ Langel, M. Zorko, Peptitergent w2x A. Bavec, A. Jureus, ´ B. Cigic, U. PD1 affects the GTPase activity of rat brain cortical membranes, Peptides 20 Ž1999. 177–184. w3x C. Behl, J.B. Davis, R. Lesley, D. Schubert, Hydrogen peroxide mediates amyloid b protein toxicity, Cell 77 Ž1994. 817–827. w4x E. Broulliet, A. Trembleau, D. Galanaud, M. Volovitcht, C. Bouillot, C. Valenza, A. Prochiantz, B. Allinquant, The amyloid precursor protein interacts with Go heterotrimeric protein within a cell compartment specialized in signal transduction, J. Neurosci. 19 Ž1990. 1717–1727. w5x B.L. Brown, R.P. Ekins, J.D. Albano, Saturation assay for cyclic AMP using endogenous binding protein, Adv. Cyclic Nucleotide Res. 2 Ž1972. 25–40. w6x D. Burdick, B. Soreghan, M. Know, J. Kosmoski, M. Knauer, A. Henschen, J. Yates, C. Cotman, C. Glabe, Assembly and aggregation properties of synthetic Alzheimer’s A4rb amyloid peptide analogs, J. Biol. Chem. 267 Ž1992. 546–554. w7x D.A. Butterfield, K. Hensley, M. Harris, M.P. Mattson, J.M. Carney, b-amyloid peptide free radical fragments initiate synaptosomal lipoperoxidation in a sequence-specific fashion: implications to Alzheimer’s disease, Biochem. Biophys. Res. Commun. 200 Ž1994. 710–715. w8x D.A. Butterfield, b-Amyloid-generated free radical oxidative stress and neurotoxicity: implications for Alzheimer’s disease, Chem. Res. Toxicol. 10 Ž1997. 518–526. w9x D. Cassel, Z. Selinger, Catecholamine stimulated GTPase activity in turkey erythrocyte membranes, Biochim. Biophys. Acta 452 Ž1976. 538–551. w10x S.I. Dikalov, M.P. Vitek, K.R. Maples, R.P. Mason, Amyloid b peptides do not form peptide-derived free radicals spontaneously, but can enhance metal-catalysed oxidation of hydroxylamines to nitroxides, J. Biol. Chem. 274 Ž1999. 9392–9399. w11x G. Ferrari-DiLeo, D.C. Mash, D.D. Flynn, Attenuation of muscarinic receptor–G-protein interaction in Alzheimer disease, Mol. Chem. Neuropathol. 24 Ž1995. 69–91. w12x K. Furukawa, Y. Abe, N. Akaike, Amyloid b protein-induced irreversible current in rat cortical neurones, NeuroReport 5 Ž1994. 2016–2018. w13x S.J. Gibbs, C.S. Johnson Jr., A PFG NMR experiment for accurate diffusion and flow studies in the presence of Eddy currents, J. Magn. Reson. 93 Ž1991. 395–402. w14x T. Grady, M. Fickova, H.S. Tager, D. Trivedi, J. Hruby, Stimulation and inhibition of cAMP accumulation by glucagon in canine hepatocytes, J. Biol. Chem. 262 Ž1987. 15514–15520. w15x K.E. Gridely, P.S. Green, J.W. Simpkins, A novel, synergistic interaction between 17-beta-estradiol and glutathione in the protection of neurons against beta-amyloid Ž25–35.-induced toxicity in vitro, Mol. Pharmacol. 54 Ž1998. 874–880. w16x J.D. Harper, P.T. Lansbury, Models of amyloid seeding in Alzheimers’s disease and Scrapie: Mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins, Annu. Rev. Biochem. 66 Ž1997. 385–407. w17x K. Hensley, J.M. Carney, M.P. Mattson, M. Aksenova, M. Harris, J.F. Wu, R.A. Floyd, D.A. Butterfield, A model for b-amyloid
w20x
w21x
w22x
w23x
w24x
w25x w26x
w27x w28x
w29x
w30x
w31x
w32x
w33x
w34x
w35x
187
aggregation and neurotoxicity based on free radical generated by the peptide: relevance to Alzheimer disease, Proc. Natl. Acad. Sci. U. S. A. 91 Ž1994. 3270–3274. T. Higashijima, S. Uzu, T. Nakajima, E.M. Ross, Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins ŽG proteins., J. Biol. Chem. 263 Ž1988. 6491–6494. H.M. Huang, G.E. Gibson, Altered beta-adrenergic receptor-stimulated cAMP formation in cultured skin fibroblasts from Alzheimer donors, J. Biol. Chem. 268 Ž1993. 14616–14621. X. Huang, C.S. Atwood, M.A. Hartshorn, G. Multhaup, L.E. Goldstein, R.C. Scarpa, M.P. Cuajungco, D.N. Gray, J. Lim, R.D. Moir, R.E. Tanzi, A.I. Bush, The Ab peptide of Alzheimer’s disease directly produces hydrogen peroxide through metal ion reduction, Biochemistry 38 Ž1999. 7609–7616. L.L. Iversen, R.J. Mortishire-Smith, S.J. Pollack, M.S. Shearman, The toxicity in vitro of beta-amyloid protein, Biochem. J. 311 Ž1995. 1–16. J.F. Kelly, K. Furukawa, S.W. Barger, M.R. Rengen, R.J. Mark, E.M. Blanc, G.S. Roth, M.P. Mattson, Amyloid beta-peptide disrupts carbachol-induced muscarinic cholinergic signal transduction in cortical neurons, Proc. Natl. Acad. Sci. U. S. A. 93 Ž1996. 6753–6758. T. Koppal, R. Subramaniam, J. Drake, M.R. Prasad, H. Dhillon, D.A. Butterfield, Vitamin E protects against Alzheimer’s amyloid peptide Ž25–35.-induced changes in neocortical synaptosomal membrane lipid structure and composition, Brain Res. 786 Ž1998. 270– 273. ¨ Langel, T. Land, T. Bartfai, Design of chimeric peptide ligands to U. galanin receptors and substance P receptors, Int. J. Pept. Protein Res. 39 Ž1992. 516–522. L.G. Longsworth, The mutual diffusion of light and heavy water, J. Phys. Chem. 64 Ž1960. 1914–1917. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 Ž1951. 265–275. H. Ma, C.A. Weiss, In vitro analysis of G-protein functions, Methods Cell Biol. 49 Ž1951. 471–485. I. Magnaldo, J. Pouyssegur, S. Paris, Thrombin exerts a dual effect on stimulated adenylate cyclase in hamster fibroblasts, an inhibition via a GTP-binding protein and potentiation via activation of protein kinase C, Biochem. J. 253 Ž1988. 711–719. R.J. Mark, Z. Pang, J.W. Geddes, K. Uchida, M.P. Mattson, Amyloid b-peptide impairs glucose transport in hippocampal and cortical neurons: involvement of membrane lipid peroxidation, J. Neurosci. 17 Ž1997. 1046–1054. R.P. Mason, R.F. Jacob, M.F. Walter, P.E. Mason, N.A. Avdulov, S.V. Chochina, U. Igbavboa, W.G. Wood, Distribution and fluidizing action of soluble and aggregated amyloid b-peptide in rat synaptic plasma membranes, J. Biol. Chem. 274 Ž1999. 18801– 18807. M.P. Mattson, J.G. Begley, R.J. Mark, K. Furukawa, Ab25-35 induced rapid lysis of red blood cells: contrast with Ab1–42 and examination of underlying mechanisms, Brain Res. 771 Ž1997. 147–153. F.R. McKenzie, Basic techniques to study G-protein function, in: G. Milligan ŽEd.., Signal Transduction. A Practical Approach, Oxford University Press, Oxford, 1992, pp. 31–56. M. Mousli, J.-L. Bueb, C. Bronner, B. Rouot, Y. Landry, G protein activation: a receptor-independent mode of action for cationic amphiphilic neuropeptides and venom peptides, Trends Pharmacol. Sci. 11 Ž1990. 358–362. I. Nishimoto, T. Okamoto, Y. Matsuura, S. Takahashi, T. Okamoto, Y. Murayama, E. Ogata, Alzheimer amyloid protein precursor complexes with brain GTP-binding protein GŽo., Nature 362 Ž1993. 75–79. T. Okamoto, S. Takeda, Y. Murayama, E. Ogata, I. Nishimoto,
188
w36x
w37x
w38x
w39x
w40x
w41x
w42x
w43x
w44x
w45x
U. Soomets et al.r Brain Research 850 (1999) 179–188 Ligand-dependent G protein coupling function of amyloid transmembrane precursor, J. Biol. Chem. 270 Ž1995. 4205–4208. B.M. Ross, M. McLaughlin, M. Roberts, G. Milligan, J. McCulloch, J.T. Knowler, Alterations in the activity of adenylate cyclase and high affinity GTPase in Alzheimer’s disease, Brain Res. 622 Ž1993. 35–42. A. Schnecko, K. Witte, J. Bohl, T. Ohm, B. Lemmer, Adenylyl cyclase activity in Alzheimer’s disease brain: stimulatory and inhibitory signal transduction pathways are differently affected, Brain Res. 644 Ž1994. 291–296. D. Schubert, C. Behl, R. Lesley, A. Brack, R. Dargusch, Y. Sagara, H. Kimura, Amyloid peptides are toxic via a common oxidative mechanism, Proc. Natl. Acad. Sci. U. S. A. 92 Ž1995. 1989–1993. S. Sheriff, A. Balasubramaniam, Inhibitory and stimulatory effects of neuropeptide Y Ž17–36. on rat cardiac adenylate cyclase activity, J. Biol. Chem. 267 Ž1992. 4680–4685. Y. Shin, R.W. Moni, J.E. Lueders, J.W. Daly, Effects of amphiphilic peptides mastoparan and adenoregulin on receptor binding, G-proteins, phosphoinositide breakdown, cyclic AMP generation, and calcium influx, Cell. Mol. Neurobiol. 14 Ž1994. 133–157. A. Smine, X. Xu, K. Nishiyama, T. Katada, P. Gambetti, S.P. Yadav, X. Wu, Y.-C. Shi, S. Yasuhara, V. Homburger, T. Okamoto, Regulation of brain G-protein Go by Alzheimer’s disease gene presenilin-1, J. Biol. Chem. 273 Ž1998. 16281–16288. C. Soto, E.M. Sigurdsson, L. Morelli, R.A. Kumar, E.M. Castano, B. Frangione, b-Sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: Implications for Alzheimer’s therapy, Nature Medicine 4 Ž1998. 822–826. E.O. Stejskal, J.E. Tanner, Spin diffusion measurements: spin echoes in the presence of a time-dependent field gradient, J. Chem. Phys. 42 Ž1965. 288–292. E. Terzi, G. Holzemann, J. Seelig, Self-association of beta-amyloid peptide Ž1–40. in solution and binding to lipid membranes, J. Mol. Biol. 252 Ž1995. 633–642. L.O. Tjernberg, J. Naslund, F. Lindqvist, J. Johansson, A.R. ¨ Karlstrom, ¨ J. Thyberg, L. Terenius, C. Nordstedt, Arrest of b-
w46x
w47x
w48x
w49x
w50x
w51x
w52x
w53x
amyloid fibril formation by a pentapeptide ligand, J. Biol. Chem. 271 Ž1996. 8545–8548. L.O. Tjernberg, C. Lilliehook, S. Hahne, ¨¨ J.E. Callaway, J. Naslund, ¨ J. Thyberg, L. Terenius, C. Norstedt, Controlling amyloid b-peptide fibril formation with protease-stable ligands, J. Biol. Chem. 272 Ž1997. 12601–12605. J.O. Tjernberg, J.E. Callaway, A. Tjernberg, S. Hahne, C. Lillehook, ¨¨ L. Terenius, J. Thyberg, C. Nordstedt, A molecular model of Alzheimer’s amyloid b-peptide fibril formation, J. Biol. Chem. 274 Ž1999. 12619–12625. J. Ulas, L.C. Brunner, L. Nguyen, C.W. Cotman, Reduced density of adenosine A1 receptors and preserved coupling of adenosine A1 receptors to G proteins in Alzheimer hippocampus: a quantitative autoradiographic study, Neuroscience 52 Ž1993. 843–854. ¨ Langel, A. Valkna, A. Jureus, ´ E. Karelson, M. Zilmer, T. Bartfai, U. Differential regulation of adenylate cyclase activity in rat ventral and dorsal hippocampus by rat galanin, Neurosci. Lett. 187 Ž1995. 75–78. W. Xia, J. Zhang, D. Kholodenko, M. Citron, M.B. Podlisny, D.B. Teplow, C. Haas, P. Seubert, E.H. Koo, D.J. Selkoe, Enhanced production and oligomerization of the 42-residue amyloid b-protein by chinese hamster ovary cells stably expressing mutant presenilins, J. Biol. Chem. 272 Ž1997. 7977–7982. S.M. Yatin, M. Aksenov, D.A. Butterfield, The antioxidant vitamin E modulates amyloid b-peptide induced creatine kinase activity inhibition and increased protein oxidation: implications for the free radical hypothesis of Alzheimer’s disease, Neurochem. Res. 24 Ž1999. 427–435. S.M. Yatin, M. Yatin, T. Aulick, K.B. Ain, D.A. Butterfield, Alzheimer’s amyloid b-peptide associated free radicals increase rat embryonic neuronal polyamine uptake and ornithine decarboxylase activity: protective effect of vitamin E, Neurosci. Lett. 263 Ž1999. 17–20. ¨ Langel, Differential M. Zorko, M. Pooga, K. Saar, K. Rezaei, U. regulation of GTPase activity by mastoparan and galparan, Arch. Biochem. Biophys. 349 Ž1998. 321–328.
Research report
Regulation of GTPase and adenylate cyclase activity by amyloid b-peptide and its fragments in rat brain tissue Ursel Soomets a,b , Riina Mahlapuu b , Roya Tehranian a , Juri ¨ Jarvet c , Ello Karelson b , b a a,d c ¨ Mihkel Zilmer , Kerstin Iverfeldt , Matjaz Zorko , Astrid Graslund , Ulo Langel a, ) ¨ a
Department of Neurochemistry and Neurotoxicology, Arrhenius Laboratories, Stockholm UniÕersity, S-106 91 Stockholm, Sweden b Department of Biochemistry, Tartu UniÕersity, EE2400 Tartu, Estonia c Department of Biophysics, Stockholm UniÕersity, S-106 91 Stockholm, Sweden d Institute of Biochemistry, Medical Faculty, UniÕersity of Ljubljana, VrazoÕ trg 2, 1000 Ljubljana, SloÕenia Accepted 21 September 1999
Abstract Modulation of GTPase and adenylate cyclase ŽATP pyrophosphate-lyase, EC 4.6.1.1. activity by Alzheimer’s disease related amyloid b-peptide, AbŽ1–42., and its shorter fragments, AbŽ12–28., AbŽ25–35., were studied in isolated membranes from rat ventral hippocampus and frontal cortex. In both tissues, the activity of GTPase and adenylate cyclase was upregulated by AbŽ25–35., whereas AbŽ12–28. did not have any significant effect on the GTPase activity and only weakly influenced adenylate cyclase. AbŽ1–42., similar to AbŽ25–35., stimulated the GTPase activity in both tissues and adenylate cyclase activity in ventral hippocampal membranes. Surprisingly, AbŽ1–42. did not have a significant effect on adenylate cyclase activity in the cortical membranes. At high concentrations of AbŽ25–35. and AbŽ1–42., decreased or no activation of adenylate cyclase was observed. The activation of GTPase at high concentrations of AbŽ25–35. was pertussis toxin sensitive, suggesting that this effect is mediated by GirGo proteins. Addition of glutathione and N-acetyl-L-cysteine, two well-known antioxidants, at 1.5 and 0.5 mM, respectively, decreased AbŽ25–35. stimulated adenylate cyclase activity in both tissues. Lys-AbŽ16–20., a hexapeptide shown previously to bind to the same sequence in Ab-peptide, and prevent fibril formation, decreased stimulation of adenylate cyclase activity by AbŽ25–35., however, NMR diffusion measurements with the two peptides showed that this effect was not due to interactions between the two and that AbŽ25–35. was active in a monomeric form. Our data strongly suggest that Ab and its fragments may affect G-protein coupled signal transduction systems, although the mechanism of this interaction is not fully understood. q 1999 Elsevier Science B.V. All rights reserved. Keywords: GTPase; Adenylate cyclase; Amyloid b-peptide; Alzheimer’s disease
1. Introduction In several recent reports, the alteration of the cellular signal transduction system in the brain of patients with the Alzheimer’s disease ŽAD. has been pointed out. It has been shown w11,36,37x that the overall activity of G-proteins is altered in the post-mortem brain of AD patients. Impaired coupling of adenosine A1 receptors w48x, badrenergic receptors w19x and other receptors to G-proteins in different tissues such as brain and skin of AD patients has been reported. The reasons for such alterations in cellular signal transduction in brain of AD patients are not )
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clarified yet, although the change in parameters of oxidative stress has been suggested as one of the possible causes w23x. Various fragments of the amyloid precursor protein, APP, affect important functions in the brain. The part of the intracellular C-terminus of APP Žsequence 657–676. has been shown to interact with brain Go-proteins w34x. It has been suggested that abnormal APP-Go signalling can be involved in the AD process and that APP has a potential receptor function w35x. Recently, Brouillet et al. w4x showed the regulation of Go GTPase activity by APP in a caveolae-like compartments specialised in signal transduction. Also, Smine et al. w41x have demonstrated that presenilin-1, a product of the familial AD gene, interacts with brain Go proteins. The described specific interactions of
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 1 4 2 - 3
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APP with presenilins w50x, may play a significant role in intramembraneous proteolysis of APP as well as of abnormal signal transduction in early onset AD ŽFAD.. A 39–43 amino acids long proteolytic fragment of APP, amyloid b-peptide ŽAb ., is a major component of the senile plaques associated with AD. The sequences of AbŽ1–42. and the peptides derived from it used in the present study are shown in Fig. 1. The most toxic fragments of Ab, AbŽ25–35. and AbŽ12–28., modulate neuronal function and immune and inflammatory responses in several cell types w36,37x. Although these peptides have not been found as naturally occurring degradation products of APP, they are widely used in studies of the mechanisms of action of Ab in in vitro studies. AbŽ1–40. and AbŽ25– 35. peptides both disrupt carbachol-induced M1 muscarinic cholinergic signal transduction in cortical neurons w22x suggesting that Ab peptides interfere with muscarinic receptor coupling to G-proteins. These results indicate that Ab plays an important role in the impairment of cholinergic transmission that occurs in AD, probably with the involvement of free radicals in the mechanism w22,38x. It has been suggested that the toxicity of Ab and its fragments is well-correlated with the degree of aggregation of these peptides. The aggregation of AbŽ1–40. and Ž1–42. peptides into fibrils has been shown to depend on pH, concentration, and the incubation time in aqueous solutions in vitro w6,16x. The polymerisation of full-length Ab into b-amyloid fibrils could be inhibited by the pentapeptide AbŽ16–20. with the sequence KLVFF as well as with its better water soluble analogue Lys-AbŽ16–20., KKLVFF w45–47x. The KLVFF-sequence is the region in Ab that most efficiently binds to Ab, and is necessary for fibril formation w45x. The mechanism of interaction of the Ab peptide with possible different signal transduction routes is, however, not clear today. Due to the cationicr amphiphilic nature of Ab-peptides, which may allow them to interact with and penetrate biological membranes w12,30x, it is not excluded that their action is mediated via G-proteins, i.e., via a receptor-independent mode. In the neurons the mechanism of neurotoxicity of Ab fragments appears also to involve generation of free radicals, induction of reactive oxygen species and to cause
lipid peroxidation w3,7,8,51,52x. Different reports have shown cellular release of peroxides, superoxide and nitric oxide in response to Ab and its fragments treatment w3,20x. Spontaneous generation of free radicals by AbŽ1–40. and AbŽ25–35. themselves in free-cell system has been reported w17x. In contrast, the recent study by Dikalov et al. w10x showed that those peptides do not yield radical adducts in the same system. It is shown that AbŽ1–40. and AbŽ25–35. can have similar or different cytotoxic mechanisms in different cell types w31x. Interactions of stimulated 7TM receptors with the asubunits of G-proteins result in alteration of GTPase activity leading to different cellular events, such as the synthesis of second messengers, e.g., cyclic AMP, generated by a membranous enzyme adenylate cyclase. It has been shown that several short amphiphilic peptides are able to directly affect the functions of G-proteins w2,33,40,53x in this way mimicking the action of native membrane proteins. In the present study, we have examined the influence of the APP-derived peptides, AbŽ25–35., AbŽ12–28., and AbŽ1–42. on the activity of GTPase and adenylate cyclase in membranes from rat ventral hippocampus and cerebral cortex. The influence of two well-known antioxidants on the enzymatic activity of adenylate cyclase was characterised as well. Since the Ab-related toxicity is associated with an aggregated state of the peptide, we studied the possible aggregation of the fragments AbŽ25–35. and AbŽ12–28. by diffusion measurements using NMR. The possible interaction of Lys-AbŽ16–20., a motif in Ab peptide essential for Ab–Ab fibril formation and binding w45,46x, with AbŽ25–35. was studied by the same method, since we show that the modulation of GTPase and adenylate cyclase activity by AbŽ25–35. was altered by LysAbŽ16–20.. As control, the effect of scrambled AbŽ25–35. on the activity of GTPase and adenylate cyclase was tested. 2. Materials and methods 2.1. Materials Amino acid derivatives were purchased from Nova Chemical, UK. Tritiated cyclic AMP Ž59 Cirmmol. was obtained from Amersham, UK. w g-32 PxGTP Ž5000 Cirmmol. was from NEN research products, UK. Na 2 ATP, GTP, cyclic AMP, phosphoenol pyruvate, bacitracin, bovine serum albumin, glutathione, N-acetyl-L-cysteine and pertussis toxin ŽPTX. were from Sigma, USA, or Fluka, Switzerland. All reagents used were of highest analytical grade. AbŽ1–42. was purchased from Bachem, Switzerland. 2.2. Peptide synthesis
Fig. 1. Amino acid sequences of AbŽ1–42., AbŽ12–28., Lys-AbŽ16–20., AbŽ25–35., and AbŽ25–35.-scrambled peptides.
All the shorter synthetic fragments of Ab peptides were synthesized in a stepwise manner in a 0.1-mmol scale on
U. Soomets et al.r Brain Research 850 (1999) 179–188
an Applied Biosystem Model 431A peptide synthesiser on a solid support using N, N X-dicyclohexylcarbodiimidehydroxybenzotriazole activation strategy. tert-Butyloxycarbonyl amino acids were coupled as hydroxybenzotriazole esters to a phenylacetamidomethyl-resin Ž0.6 mmol amino groups per gram resin, Novabiochem, Switzerland. to achieve the C-terminal free carboxylic acids. The peptides were finally cleaved from the resin with liquid HF at 08C for 30 min. Deprotection of the side chains, cleavage of the peptides and purification on HPLC have been described in detail earlier w24x. The purity of the peptides was ) 99% as demonstrated by HPLC on an analytical Nucleosil 120-3 C 18 reversed-phase column Ž0.4 cm = 10 cm.. The molecular masses of the peptides were determined by a plasma desorption mass spectrometry ŽBioion 20, Applied Biosystems. and the calculated values were obtained in each case. 2.3. Preparation of peptide solutions Lyophilised peptides were freshly resuspended in the assay buffer at room temperature, diluted and assayed immediately for adenylate cyclase and GTPase activity measurements. 2.4. GTPase assay Measurement of GTPase activity was performed radio metrically according to Cassel and Selinger w9x, with the modifications suggested by McKenzie w32x. Membranes from rat brain frontal cortex and hippocampus were obtained according to the protocol of McKenzie w32x with minor modifications as described previously w53x. To the diluted membranes Žfinal protein concentration in the assay mixture was 0.2 mgrml, as determined by the method of Lowry et al. w26x. the ice-cold reaction cocktail containing ATP Ž1 mM., 5X-adenylylimido-diphosphate Ž1 mM., ouabain Ž1 mM., phosphocreatine Ž10 mM., creatine phosphokinase Ž2.5 Unitsrml., dithiothreitol Ž4 mM., MgCl 2 Ž5 mM., NaCl Ž100 mM., and trace amounts of w g-32 PxGTP to give 50.000–100.000 c.p.m. in an aliquot of the reaction cocktail Žwith the addition of cold GTP to give the required total concentration of GTP of 0.5 mM. was added. Incubation medium was standard TE-buffer Ž10 mM Tris– HCl q 0.1 mM EDTA., pH 7.5. Background low-affinity hydrolysis of w g-32 PxGTP was assessed by incubating parallel tubes in the presence of 100 mM GTP. Blank values were determined by the replacement of the membrane solution with assay buffer. GTPase reaction was started by transferral of the reaction mixtures to 308C water bath for 12 min. Unreacted GTP was removed by the 5% suspension of the activated charcoal in 20 mM H 3 PO4 . The radioactivity of the released radioactive phosphate was determined in the LKB 1214 Rackbeta or in Packard 3255
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liquid scintillation counter. The basal GTPase activity of the rat brain cortical membrane preparation was 65 " 3 pmolrminrmg protein and that of the ventral hippocampal membranes was 52 " 4 pmolrminrmg protein. 2.5. Adenylate cyclase assay Membranes of ventral hippocampus and frontal cortex were prepared from Wistar rats Ž200–300 g., according to previously published procedures w49x. Homogenates Žin 8 mM HEPES-Na, pH 7.4. of precooled ventral hippocampus were diluted, stirred on ice for 30 min and centrifuged for 6 min at 1600 = g. The pellets were resuspended in ice-cold protein-buffer Ž4 mM HEPES-Na, 1.5 mM theophylline, 8.25 mM MgCl 2 , 0.75 mM EGTA, 7.5 mM KCl, 100 mM NaCl, pH 7.4. to a final protein concentration of 0.6–0.8 mgrml. The basal adenylate cyclase activity was assayed at 0.04 mgrml of membrane protein in reactionbuffer, additionally containing Žin protein buffer. 100 mgrml bacitracin, 0.03% bovine serum albumin, 10 mM phosphoenol-pyruvate and 30 mgrml pyruvate kinase w49x. In all experiments, the peptides, dissolved in the reaction buffer, were added to the assay mixture 2 min before the reaction was initiated by 10 mM ATPr10 mM GTP. The reaction at 308C was terminated after 15 min by the addition of 100 mM EDTA, followed by boiling the samples for 3 min. The cyclic AMP content in the tubes was measured by a competitive protein saturation assay using cyclic AMP-binding protein from bovine adrenal cortex w5x. The basal levels of the adenylate cyclase activity in ventral hippocampus and in frontal cortex were 52 " 3 and 76 " 1 pmol cyclic AMPrminrmg protein, respectively. The protein content of the membrane preparations was determined according to Lowry et al. w26x. 2.6. Incubation of tissue membranes with antioxidants We have examined the effects of the antioxidants, glutathione and N-acetyl-L-cysteine, on the basal activity of adenylate cyclase as well antioxidant induced alterations in the modulation of adenylate cyclase activity by AbŽ25– 35.. The effect was measured as a difference in the amount of cAMP, produced by membranous adenylate cyclase in the presence or absence of 10y7 M AbŽ25–35. and in the conditions where glutathione Žfinal concentration 1.5 mM. or N-acetyl-L-cysteine Ž0.5 mM. were added to the medium before the peptide, and shown as percentage of changes in cAMP production against unaffected Žbasal. activity Žs 100%.. 2.7. PTX catalysed ADP-ribosylation PTX was activated by treatment with 50 mM dithiotreitol ŽDTT. for 30 min at 378C. For the ADP-ribosylation reaction, the membranes from brain cortex and ventral
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hippocampus were treated with 10 mgrml of PTX in the 10 mM HEPES-Na or TE buffer ŽpH 7.4., containing 3 mM NADq, 20 mM thymidine, 1 mM ATP and 100 mM GTP w27x. The treatment was initiated by transferring the tissue membranes to a 378C water bath for 30 min. After the incubation, the membranes were diluted two-fold and centrifuged at 10 000 = g for 45 min. The obtained pellet was resuspended with ice-cold buffer and used for the GTPase or adenylate cyclase assay experiments.
Table 1 Translational diffusion coefficients of Ab fragments Lys-AbŽ16–20. and AbŽ25–35., determined using pulsed field gradient NMR spectroscopy Experimental values of the translational diffusion coefficient D along with calculated molecular weights are listed with theoretical molecular weights. Samples used were 300 mM peptide in 70 mM phosphate buffer, pH 7.0, at 258C.
Lys-AbŽ16–20. AbŽ25–35.
10 6 Drcm2 sy1
Molecular weight rDa
Molecular weight Žtheor.rDa
3.13"0.05 2.76"0.05
760"50 1230"100
723.9 1063.3
2.8. Measurements of translational diffusion coefficients by NMR NMR spectra were acquired on a Varian Inova 600 MHz spectrometer at 298 K. Peptide samples were prepared in 70 mM phosphate buffer, pH 7.0, with concentrations ranging from 300 mM to 1 mM. The translational diffusion coefficient was measured using pulsed field gradient longitudinal eddy-current delay type pulse sequence w13,43x. The gradient strength was calibrated using a HDO signal in 99.9% D 2 O and literature value for diffusion coefficient of 1.9 = 10y5 cm2 sy1 w25x. In the diffusion studies, the integral of the methyl resonances between 0.97 and 0.71 ppm was used for the Lys-AbŽ16–20. peptide and the integral of the Met methyl signal at 2.10 ppm was used for the AbŽ25–35. fragment. In diluted samples the diffusion coefficient D is related to the molecular hydrodynamic radius R h according to the Stokes-Einstein law: D s k B Tr6ph R h , where k B is the Boltzmann constant, T is the absolute temperature, and h is the viscosity of water. From the known specific volume of proteins, V s 0.73 cm3 gy1 and assuming a hydration ˚ one can calculate the molecular weight layer rw s 1.8 A, of a peptide Mw from the experimentally determined hydrodynamic radius R h : Mw s Ž4r3. p Ž R h y rw . 3 NAVy1. Translational diffusion coefficients of Ab fragments LysAbŽ16–20. and AbŽ25–35. are shown in Table 1.
3. Results
3.1. GTPase actiÕity The effects of Ab peptides on the activity of GTPase in membranes isolated from rat ventral hippocampus and frontal cortex are illustrated in Fig. 2. In membranes from both brain regions, the GTPase activity was stimulated by AbŽ25–35. peptide and increased by 95% and 104%, respectively, at 100 mM concentration ŽFig. 2A and B.. The EC 50 value of the activation was 29 " 4 mM in the ventral hippocampal membranes Ž n H s 1.7 " 0.3. and 26
" 3 mM Ž n H s 2.4 " 0.7. in the cortical membranes. In contrast, AbŽ25–35. peptide with the scrambled sequence ŽFig. 1. used as control at the all tested concentrations Ž10y9 to 10y5 M. had no effect on GTPase activity Ždata not shown.. The effects of AbŽ1–42. on GTPase activity from both brain regions were also investigated and are shown in Fig. 2. The EC 50 value of the stimulatory effect of AbŽ1–42. was 1.36 " 0.25 mM in the ventral hippocampal membranes and 1.33 " 0.23 mM in the cortical membranes. The concentration-dependent effect of AbŽ1– 42. on GTPase activity was bell-shaped and declined at higher concentrations of the peptide. AbŽ12–28. had no effect on GTPase activity in the concentrations tested. The hexapeptide, Lys-AbŽ16–20., which had no effect on its own, slightly decreased the maximal effect of AbŽ25–35. to 60% and 75% in the ventral hippocampal and cortical membranes, respectively, leaving the values of EC 50 and n H practically unchanged. PTX catalysed ribosylation of the membranes totally prevented activation of GTPase by AbŽ25–35. peptide ŽFig. 2A and B., suggesting that AbŽ25–35. activates GirGo but not Gs type of G-proteins.
3.2. Adenylate cyclase actiÕity In Fig. 3A and B, the effects of AbŽ25–35., AbŽ1–42., AbŽ12–28. and Lys-AbŽ16–20. on the activity of adenylate cyclase in the membranes from rat ventral hippocampus and frontal cortex are presented. In the membranes from both brain regions, AbŽ25–35. exhibited a bellshaped dose-dependence effect on the activity of adenylate cyclase. In ventral hippocampus ŽFig. 3A., AbŽ25–35. stimulated the enzyme at the lower concentrations with the maximal value of 33 " 2% at 0.1 mM ŽEC 50 s 1.7 nM.. At higher concentrations of AbŽ25–35. Žup to 10 mM., no stimulatory effect of the peptide could be observed. In the frontal cortical membranes ŽFig. 3B., the dose–response curve for AbŽ25–35. was similar ŽEC 50 s 2.7 nM. to that in the ventral hippocampus, but the maximal activation was lower Ž23 " 7% at 0.1 mM.. The activity of adenylate cyclase was not affected by scrambled AbŽ25–35. peptide
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Fig. 2. Effects of AbŽ25–35. Žv ., AbŽ1–42., ŽX., AbŽ12–28. Ž'., and Lys-AbŽ16–20. Ž^., on GTPase activity in rat ventral hippocampal ŽA. and frontal cortical ŽB. membranes: the effect of 100 mM Lys-AbŽ16–20. ŽI., and PTX catalysed ADP-ribosylation Žmembranes were treated at 378C with 10 mgrml PTX for 30 min. Ž`., on the activation of GTPase by AbŽ25–35. is also shown. The data represent the average of four independent experiments. S.E.M. never exceeded 10%; error bars are not shown for clarity. Curves for the activation of GTPase by AbŽ25–35. in the absence and in the presence of Lys-AbŽ16–20. were obtained by non-linear regression fitting of the single phase dose–response equation using Prism computer program ŽGraphPad, USA..
tested in the same concentrations as AbŽ25–35. Ždata not shown.. The effect of AbŽ1–42. on adenylate cyclase activity was also studied in ventral hippocampus and was similar to the effects of AbŽ25–35.. AbŽ1–42. stimulated the enzyme with the maximum value of 45 " 4% at 0.05 mM ŽEC 50 s 2 nM.. Further increase in the concentration of the peptide reversed the stimulatory effect on the enzymatic activity. In the frontal cortical membranes, AbŽ1–42. had no effect on the activity of adenylate cyclase in the concentrations tested. AbŽ12–28. slightly increased the activity of adenylate cyclase in ventral hippocampus, whereas, in the frontal cortex the effects of this peptide were not statistically significant. The hexapeptide Lys-
AbŽ16–20. had no effect on adenylate cyclase activity in ventral hippocampus and cortex. Since the hexapeptide Lys-AbŽ16–20. could decrease the stimulatory effect of AbŽ25–35. on GTPase activity ŽFig. 2., we studied whether the hexapeptide could interfere with AbŽ25–35. induced stimulation of adenylate cyclase activity. Membranes from ventral hippocampus were incubated with 0.1 mM AbŽ25–35. or AbŽ12–28. for 2 min before Lys-AbŽ16–20. Ž1 nM–10 mM. was added. These experiments demonstrate ŽFig. 5A and B. that the stimulatory effects of AbŽ25–35. on the adenylate cyclase activity in the membranes from ventral hippocampus and cortex were completely inhibited by Lys-AbŽ16– 20. at higher concentrations. This hexapeptide also re-
Fig. 3. Effects of AbŽ25–35. Žv ., AbŽ1–42. ŽX., AbŽ12–28. Ž'., and Lys-AbŽ16–20. Ž`., on adenylate cyclase activity in rat ventral hippocampal ŽA. and frontal cortical ŽB. membranes. The data represent mean " S.E.M. of three independent experiments Ž100% corresponds to the basal adenylate activity..
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Fig. 4. The effect of AbŽ25–35. on adenylate cyclase activity in the rat ventral hippocampal ŽA. and frontal cortical ŽB. membranes treated at 378C with 10 mgrml PTX for 30 min before the experiment. The data represent mean " S.E.M. of three independent experiments Ž100% corresponds to the basal adenylate activity..
versed the stimulation of adenylate cyclase activity by AbŽ12–28. in the ventral hippocampal membranes Ždata not shown.. 3.2.1. Effect of glutathione and N-acetyl-L-cysteine on adenylate cyclase actiÕity Reduced glutathione ŽGSH. has previously been shown to protect SK-N-SH human neuroblastoma cells from AbŽ25–35. toxicity w15x. We examined the effect of glutathione and N-acetyl-L-cysteine on the activity of adenylate cyclase in rat ventral hippocampal and cerebral cortical membranes. Glutathione at 1.5 mM alone inhibited the basal activity of adenylate cyclase Ž100%. in ventral hippocampal membranes by 32.8 " 5.2% and in cortical membranes by 7.4 " 3.0%. Surprisingly, N-acetyl-L-cysteine increased the adenylate cyclase basal activity in ventral hippocampus and in frontal cortex by 19.4 " 3.5% and 16.3 " 3.6%, respectively. Incubation of membranes with antioxidants and 0.1 mM AbŽ25–35. abolished or decreased the stimulation of
adenylate cyclase activity by this fragment of Ab. Glutathione at 1.5 mM, together with 0.1 mM AbŽ25–35., showed the decrease of basal enzyme activity by 22.5 " 8.3% in ventral hippocampal and 9.7 " 0.9% in cortical membranes. N-acetyl-L-cysteine at 0.5 mM decreased the AbŽ25–35. stimulated adenylate cyclase activity from 32.5 " 2.1% to 8.2 " 1.4% in ventral hippocampus and in frontal cortex from 22.6 " 7.0% to 6.0 " 1.0% over basal activity. 3.3. Effect of PTX on actiÕity of adenylate cyclase To further elucidate the mechanismŽs. behind the bellshaped dependency of the adenylate cyclase activity on the AbŽ25–35., we investigated if the effect was PTX-sensitive on membranes prepared from hippocampus and cortex. Fig. 4 shows that in PTX-treated membranes a dosedependent stimulatory effect of AbŽ25–35. was retained both in ventral hippocampus Žmaximal effect 30.7%, EC 50s s 11.1 nM. and cortex Žmaximal effect 33.2%, EC 50 s 2.55 nM., whereas no attenuation of the stimula-
Fig. 5. The effect of Lys-AbŽ16–20., on the stimulated adenylate cyclase activity. ŽA. Rat ventral hippocampal membranes stimulated by 10y7 M of AbŽ25–35.. ŽB. Rat frontal cortical membranes stimulated by 10y8 M of AbŽ25–35.. The data represent mean " S.E.M. of three independent experiments Ž100% corresponds to the basal adenylate activity..
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tion at higher concentrations Žup to 10 mM. of AbŽ25–35. could be observed. 3.4. NMR measurements The propensity of the peptides to aggregate in aqueous solution was investigated by diffusion studies using NMR. The experimentally determined translational diffusion coefficients and calculated molecular weights along with the theoretical molecular weights for Lys-AbŽ16–20. and AbŽ25–35. peptides are summarised in Table 1. The results show that both peptides have values close to that expected for a monomeric state under the conditions used, i.e., the same pH and somewhat higher concentrations than those used in the activity studies. For AbŽ12–28., under the same conditions, aggregation was immediate and to such extent that NMR-based diffusion measurements became impossible due to the large line width in the NMR spectra Ždata not shown.. Measurements were performed also at pH 5.0 for all three peptides with similar results as at pH 7.0. In the 1:1 mixture of Lys-AbŽ16–20. and AbŽ25–35., the diffusion coefficients of the two peptides were determined as 3.13 = 10y6 and 2.76 = 10y6 cm2 sy1 , respectively, i.e., unchanged from when they were measured in separate samples ŽTable 1.. The results indicate that two peptides do not interact with each other. 4. Discussion Cerebral deposition of amyloid, an insoluble extracellular precipitate mainly consisting of Ab peptide, seems to be a central event in AD. The mechanisms by which the amyloid deposition is associated with dementia in AD is rather elusive. Alterations in signal transduction mechanisms involving different G-protein coupled receptors have been shown in AD. Here, we discuss the effects of AbŽ1– 42. and its shorter fragments, AbŽ25–35. and AbŽ12–28., on the activity of two key enzymes, GTPase and adenylate cyclase, in cellular signal transduction. In membrane preparations from hippocampus and cortex, AbŽ25–35. and AbŽ1–42. stimulated the activity of membrane-bound GTPase. The maximal effect of AbŽ25– 35., observed at 100 mM concentrations, was two-fold above the basal activity of the enzyme. The maximal effect of AbŽ1–42. on GTPase activity was about 1.5-fold above basal activity and detected at much lower concentration Ž5–7 mM. of the peptide. The effect of AbŽ1–42. on the GTPase activity was bell-shaped. AbŽ1–42. stimulation of GTPase activity declined at concentrations above 5 mM both in hippocampal and cortical membranes. AbŽ1–42. activation of adenylate cyclase in hippocampal membranes was similar to that of AbŽ25–35.. AbŽ1–42. stimulated adenylate cyclase to 30%–40% above basal activity in nanomolar concentration range, however, above 100 nM concentration, the effect was attenuated. In membranes from the cerebral cortex, the
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full-length Ab peptide had no significant effects on adenylate cyclase activity. There might be several explanations for the distinct effects of AbŽ25–35. and the full-length AbŽ1–42. on the activity of adenylate cyclase and GTPase. As it has been proposed the AbŽ25–35. and the full-length peptide may activate different signalling systems and therefore have different effects on the systems used in our experiments. Another explanation for the different effects might be due to the different heterogeneity of G-proteins between these brain regions. A third explanation may lie in the fact that the two peptides may interact differently with membrane components, for example, Mason et al. w30x, showed recently that AbŽ1–42. and AbŽ1– 40. behaved differently within the membrane. AbŽ1–42. interacted only with the membrane lipid bilayer hydrocarbon core, both in aggregated and soluble form, whereas AbŽ1–40., in its aggregated form interacts with hydrated surface of the membrane and is associated with the hydrocarbon core when it is soluble. Although the stimulation of GTPase by AbŽ25–35. was similar in both tissues, the value of Hill coefficient Ž n H . for this activatory peptide was somewhat higher in the frontal cortex than in the ventral hippocampus, 2.4 and 1.7, respectively. The different heterogeneity in G-protein subunits in these tissues, the multiple binding to G-proteins or interaction between the peptide and differently composed membranes could provide an explanation for these different n H values. It has been shown previously that the accumulation, conformation and orientation of Ab peptides in cell membranes can be affected by a heterogeneous composition of membrane lipids, proteins and carbohydrates w44x. A dual response of adenylate cyclase to the neurotoxic AbŽ25–35. in membranes of rat ventral hippocampus and frontal cortex was found. This response includes a consistent, 20%–30% enhancement of adenylate cyclase activity at low concentrations and less or no effects at higher concentrations of the peptide. Such a biphasic mechanism of action at adenylate cyclase has been previously demonstrated for several neuronal hormones such as NPYŽ17–36., glucagon and thrombin w14,28,39x. Both the GirGo-proteins are present in large quantities in the mammalian brain and serve as the major contributors to the high affinity GTPase activity w1,36x. Since the significant increase of GTPase activity and the reduced stimulation of adenylate cyclase activity were detected at relatively high concentrations Ž10–100 mM. of AbŽ25–35., one could assume that the peptide inhibits adenylate cyclase at higher concentrations via activation of inhibitory GirGo-proteins. In fact, in PTX-treated membranes, AbŽ25–35. stimulation at higher concentrations was no longer attenuated ŽFig. 4.. We suggest that the inhibitory phase in the pronounced bell-shaped effect of AbŽ25–35. on the brain adenylate cyclase activity Žcf. Fig. 3A and B. may be directly mediated by low-affinity isoformŽs. of Gi-proteins. This
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suggestion is strongly corroborated by the ability of PTX to totally prevent the activation of GTPase by AbŽ25–35. both in the membranes from the frontal cortex and from the ventral hippocampus ŽFig. 2. and, additionally, by the fact that AbŽ25–35. shares some structural and amphipathic features of mastoparan w2x, a known direct activator of GirGo a-subunit w18x. One possibility would be that potent stimulation of GTPase activity by high concentrations of AbŽ25–35. is specifically associated with those Gi-isoforms that inhibit the adenylate cyclase activity in the ventral hippocampus and frontal cortex. The mechanism of the activation of adenylate cyclase at low concentrations of AbŽ25–35. thus remains to be clarified, however, our results suggest that the activation of adenylate cyclase by AbŽ25–35. is not mediated via Gs-proteins. On the other hand, the suppression of cyclic AMP-signalling pathway at higher concentrations of the peptide by the stimulation of inhibitory GirGo-proteins seems to be well-supported by our study. An interesting observation in this study is that a decrease in the AbŽ25–35. stimulated adenylate cyclase activity occurs in the presence of glutathione and N-acetylL-cysteine. The significant cutback of this activation by the free radical scavengers may show that a redox component is involved in the mechanism of stimulation of adenylate cyclase activity by AbŽ25–35.. On the other hand, AbŽ25–35. has been shown to increase intracellular levels of reactive oxygen species only at the higher concentration than used by us. Glutathione decreased and N-acetyl-L-cysteine increased basal activity of adenylate cyclase in both rat brain tissues, i.e., acting differentially: the reasons for this are not clear yet. The scavengers may influence on enzyme activity by different mechanism, glutathione, for example, through the binding to the glutamate receptors. The NMR experiments show that the effects of AbŽ25– 35. are due to monomeric form of the peptide. In contrast, no effects on regulation of GTPase and adenylate cyclase activity were found for the aggregated AbŽ12–28.. These results support the hypothesis that the regulatory effects studied here are not directly related to the general toxicity of the Ab peptide and its fragments in aggregated forms, but may be due to more specific interactions, like direct interactions with GirGo proteins, as discussed above, or interaction with other components of the signal transduction systems, yet unknown. The peptide AbŽ12–28. and the hexapeptide LysAbŽ16–20. did not show any detectable effect on the GTPase activity in the tested brain regions under the specified reaction conditions. However, the hexapeptide was capable of slightly reducing the maximal activatory effect of AbŽ25–35. on the GTPase activity in the membranes from both tissues. These results suggest that the hexapeptide could directly or indirectly interfere with the interaction of AbŽ25–35. with a-subunitŽs. of G-proteins in the membranes. Our NMR-studies demonstrate the absence of the direct interaction between Lys-AbŽ16–20.
and AbŽ25–35. in a water environment, supporting the idea of indirect and probably non-specific interference of the hexapeptide. Additionally, short peptidic ligands, capable of binding efficiently to the neurotoxic AbŽ25–35., have not been identified yet although some penta- to decapeptides have been shown previously to interact with other Ab fragments as well as with full-length AbŽ1–40. w42x. The fragment AbŽ12–28. and the hexapeptide LysAbŽ16–20. generate only a slight modification of adenylate cyclase activity. Addition of the hexapeptide to the membranes after their incubation with 0.01 mM AbŽ25– 35., leads to the concentration-dependent inhibition of the stimulatory phase in the bell-shaped regulatory effect of AbŽ25–35. on adenylate cyclase activity in both brain regions ŽFig. 5.. When the hexapeptide was added to the reaction media before the AbŽ25–35., the stimulation of adenylate cyclase activity by AbŽ25–35. in both regions was suppressed in a similar way Ždata not shown.. The inhibitory phase in the adenylate cyclase modulation by higher Ž10y5 M. concentrations of AbŽ25–35. was less affected by the hexapeptide ligand. This is in accordance with a very small effect of Lys-AbŽ16–20. on the activation of GTPase by AbŽ25–35. and also corroborates the suggestion that the inhibitory effect of higher concentration of AbŽ25–35. on adenylate cyclase activation is exerted through GirGo-proteins. The attenuation of the stimulatory phase in the biphasic effect of AbŽ25–35. on the adenylate cyclase activity in the brain membranes in the presence of the hexapeptide might be due to the interaction of the cationic hexapeptide with Ga-proteins via a receptor-independent mode. Another explanation could be the action of partly hyper-aggregated intermediates of the AbŽ25–35. disrupting the cyclic AMP synthesis and leading to dysfunction of signal transduction as observed in post-mortem AD brain w21x rather than a simultaneous and additive inhibition of adenylate cyclase by the hexapeptide and AbŽ25–35.. In summary, our data show a response of G-protein, probably GirGo , coupled cyclic AMP-signalling system to the neurotoxic AbŽ25–35. in the membranes from the studied brain regions. The mechanism of action of AbŽ25– 35. may involve different subtypes of regulatory G-proteins but also other signal transduction mechanisms. The ability of Ab and its neurotoxic fragments to initiate membrane lipid peroxidation and to enhance oxidative stress in primary neuronal cultures has recently been reported w29x. Our present data suggest, in addition to the widely proposed role of amyloid aggregation, other mechanisms of amyloid peptide interaction, such as the influence on signal transduction, in Alzheimer’s disease. Acknowledgements This work was supported by Swedish Institute Žstipend to U.S.., by Wenner-Gren Center, Stockholm, Sweden
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Žstipend to M.Z.., by Structural Joint European Project S-JEP-09270-95, and by grants from Swedish Research Council for Natural Sciences ŽNFR., from Royal Swedish Academy, and from Ministry of Science and Technology of Republic of Slovenia.
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References w1x T. Asano, H. Shinohara, R. Morshita, K. Kato, Immunochemical and immunohistochemical localization of the G-protein Gi1 in rat central nervous tissues, J. Biochem. 108 Ž1990. 988–994. ¨ Langel, M. Zorko, Peptitergent w2x A. Bavec, A. Jureus, ´ B. Cigic, U. PD1 affects the GTPase activity of rat brain cortical membranes, Peptides 20 Ž1999. 177–184. w3x C. Behl, J.B. Davis, R. Lesley, D. Schubert, Hydrogen peroxide mediates amyloid b protein toxicity, Cell 77 Ž1994. 817–827. w4x E. Broulliet, A. Trembleau, D. Galanaud, M. Volovitcht, C. Bouillot, C. Valenza, A. Prochiantz, B. Allinquant, The amyloid precursor protein interacts with Go heterotrimeric protein within a cell compartment specialized in signal transduction, J. Neurosci. 19 Ž1990. 1717–1727. w5x B.L. Brown, R.P. Ekins, J.D. Albano, Saturation assay for cyclic AMP using endogenous binding protein, Adv. Cyclic Nucleotide Res. 2 Ž1972. 25–40. w6x D. Burdick, B. Soreghan, M. Know, J. Kosmoski, M. Knauer, A. Henschen, J. Yates, C. Cotman, C. Glabe, Assembly and aggregation properties of synthetic Alzheimer’s A4rb amyloid peptide analogs, J. Biol. Chem. 267 Ž1992. 546–554. w7x D.A. Butterfield, K. Hensley, M. Harris, M.P. Mattson, J.M. Carney, b-amyloid peptide free radical fragments initiate synaptosomal lipoperoxidation in a sequence-specific fashion: implications to Alzheimer’s disease, Biochem. Biophys. Res. Commun. 200 Ž1994. 710–715. w8x D.A. Butterfield, b-Amyloid-generated free radical oxidative stress and neurotoxicity: implications for Alzheimer’s disease, Chem. Res. Toxicol. 10 Ž1997. 518–526. w9x D. Cassel, Z. Selinger, Catecholamine stimulated GTPase activity in turkey erythrocyte membranes, Biochim. Biophys. Acta 452 Ž1976. 538–551. w10x S.I. Dikalov, M.P. Vitek, K.R. Maples, R.P. Mason, Amyloid b peptides do not form peptide-derived free radicals spontaneously, but can enhance metal-catalysed oxidation of hydroxylamines to nitroxides, J. Biol. Chem. 274 Ž1999. 9392–9399. w11x G. Ferrari-DiLeo, D.C. Mash, D.D. Flynn, Attenuation of muscarinic receptor–G-protein interaction in Alzheimer disease, Mol. Chem. Neuropathol. 24 Ž1995. 69–91. w12x K. Furukawa, Y. Abe, N. Akaike, Amyloid b protein-induced irreversible current in rat cortical neurones, NeuroReport 5 Ž1994. 2016–2018. w13x S.J. Gibbs, C.S. Johnson Jr., A PFG NMR experiment for accurate diffusion and flow studies in the presence of Eddy currents, J. Magn. Reson. 93 Ž1991. 395–402. w14x T. Grady, M. Fickova, H.S. Tager, D. Trivedi, J. Hruby, Stimulation and inhibition of cAMP accumulation by glucagon in canine hepatocytes, J. Biol. Chem. 262 Ž1987. 15514–15520. w15x K.E. Gridely, P.S. Green, J.W. Simpkins, A novel, synergistic interaction between 17-beta-estradiol and glutathione in the protection of neurons against beta-amyloid Ž25–35.-induced toxicity in vitro, Mol. Pharmacol. 54 Ž1998. 874–880. w16x J.D. Harper, P.T. Lansbury, Models of amyloid seeding in Alzheimers’s disease and Scrapie: Mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins, Annu. Rev. Biochem. 66 Ž1997. 385–407. w17x K. Hensley, J.M. Carney, M.P. Mattson, M. Aksenova, M. Harris, J.F. Wu, R.A. Floyd, D.A. Butterfield, A model for b-amyloid
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w35x
187
aggregation and neurotoxicity based on free radical generated by the peptide: relevance to Alzheimer disease, Proc. Natl. Acad. Sci. U. S. A. 91 Ž1994. 3270–3274. T. Higashijima, S. Uzu, T. Nakajima, E.M. Ross, Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins ŽG proteins., J. Biol. Chem. 263 Ž1988. 6491–6494. H.M. Huang, G.E. Gibson, Altered beta-adrenergic receptor-stimulated cAMP formation in cultured skin fibroblasts from Alzheimer donors, J. Biol. Chem. 268 Ž1993. 14616–14621. X. Huang, C.S. Atwood, M.A. Hartshorn, G. Multhaup, L.E. Goldstein, R.C. Scarpa, M.P. Cuajungco, D.N. Gray, J. Lim, R.D. Moir, R.E. Tanzi, A.I. Bush, The Ab peptide of Alzheimer’s disease directly produces hydrogen peroxide through metal ion reduction, Biochemistry 38 Ž1999. 7609–7616. L.L. Iversen, R.J. Mortishire-Smith, S.J. Pollack, M.S. Shearman, The toxicity in vitro of beta-amyloid protein, Biochem. J. 311 Ž1995. 1–16. J.F. Kelly, K. Furukawa, S.W. Barger, M.R. Rengen, R.J. Mark, E.M. Blanc, G.S. Roth, M.P. Mattson, Amyloid beta-peptide disrupts carbachol-induced muscarinic cholinergic signal transduction in cortical neurons, Proc. Natl. Acad. Sci. U. S. A. 93 Ž1996. 6753–6758. T. Koppal, R. Subramaniam, J. Drake, M.R. Prasad, H. Dhillon, D.A. Butterfield, Vitamin E protects against Alzheimer’s amyloid peptide Ž25–35.-induced changes in neocortical synaptosomal membrane lipid structure and composition, Brain Res. 786 Ž1998. 270– 273. ¨ Langel, T. Land, T. Bartfai, Design of chimeric peptide ligands to U. galanin receptors and substance P receptors, Int. J. Pept. Protein Res. 39 Ž1992. 516–522. L.G. Longsworth, The mutual diffusion of light and heavy water, J. Phys. Chem. 64 Ž1960. 1914–1917. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 Ž1951. 265–275. H. Ma, C.A. Weiss, In vitro analysis of G-protein functions, Methods Cell Biol. 49 Ž1951. 471–485. I. Magnaldo, J. Pouyssegur, S. Paris, Thrombin exerts a dual effect on stimulated adenylate cyclase in hamster fibroblasts, an inhibition via a GTP-binding protein and potentiation via activation of protein kinase C, Biochem. J. 253 Ž1988. 711–719. R.J. Mark, Z. Pang, J.W. Geddes, K. Uchida, M.P. Mattson, Amyloid b-peptide impairs glucose transport in hippocampal and cortical neurons: involvement of membrane lipid peroxidation, J. Neurosci. 17 Ž1997. 1046–1054. R.P. Mason, R.F. Jacob, M.F. Walter, P.E. Mason, N.A. Avdulov, S.V. Chochina, U. Igbavboa, W.G. Wood, Distribution and fluidizing action of soluble and aggregated amyloid b-peptide in rat synaptic plasma membranes, J. Biol. Chem. 274 Ž1999. 18801– 18807. M.P. Mattson, J.G. Begley, R.J. Mark, K. Furukawa, Ab25-35 induced rapid lysis of red blood cells: contrast with Ab1–42 and examination of underlying mechanisms, Brain Res. 771 Ž1997. 147–153. F.R. McKenzie, Basic techniques to study G-protein function, in: G. Milligan ŽEd.., Signal Transduction. A Practical Approach, Oxford University Press, Oxford, 1992, pp. 31–56. M. Mousli, J.-L. Bueb, C. Bronner, B. Rouot, Y. Landry, G protein activation: a receptor-independent mode of action for cationic amphiphilic neuropeptides and venom peptides, Trends Pharmacol. Sci. 11 Ž1990. 358–362. I. Nishimoto, T. Okamoto, Y. Matsuura, S. Takahashi, T. Okamoto, Y. Murayama, E. Ogata, Alzheimer amyloid protein precursor complexes with brain GTP-binding protein GŽo., Nature 362 Ž1993. 75–79. T. Okamoto, S. Takeda, Y. Murayama, E. Ogata, I. Nishimoto,
188
w36x
w37x
w38x
w39x
w40x
w41x
w42x
w43x
w44x
w45x
U. Soomets et al.r Brain Research 850 (1999) 179–188 Ligand-dependent G protein coupling function of amyloid transmembrane precursor, J. Biol. Chem. 270 Ž1995. 4205–4208. B.M. Ross, M. McLaughlin, M. Roberts, G. Milligan, J. McCulloch, J.T. Knowler, Alterations in the activity of adenylate cyclase and high affinity GTPase in Alzheimer’s disease, Brain Res. 622 Ž1993. 35–42. A. Schnecko, K. Witte, J. Bohl, T. Ohm, B. Lemmer, Adenylyl cyclase activity in Alzheimer’s disease brain: stimulatory and inhibitory signal transduction pathways are differently affected, Brain Res. 644 Ž1994. 291–296. D. Schubert, C. Behl, R. Lesley, A. Brack, R. Dargusch, Y. Sagara, H. Kimura, Amyloid peptides are toxic via a common oxidative mechanism, Proc. Natl. Acad. Sci. U. S. A. 92 Ž1995. 1989–1993. S. Sheriff, A. Balasubramaniam, Inhibitory and stimulatory effects of neuropeptide Y Ž17–36. on rat cardiac adenylate cyclase activity, J. Biol. Chem. 267 Ž1992. 4680–4685. Y. Shin, R.W. Moni, J.E. Lueders, J.W. Daly, Effects of amphiphilic peptides mastoparan and adenoregulin on receptor binding, G-proteins, phosphoinositide breakdown, cyclic AMP generation, and calcium influx, Cell. Mol. Neurobiol. 14 Ž1994. 133–157. A. Smine, X. Xu, K. Nishiyama, T. Katada, P. Gambetti, S.P. Yadav, X. Wu, Y.-C. Shi, S. Yasuhara, V. Homburger, T. Okamoto, Regulation of brain G-protein Go by Alzheimer’s disease gene presenilin-1, J. Biol. Chem. 273 Ž1998. 16281–16288. C. Soto, E.M. Sigurdsson, L. Morelli, R.A. Kumar, E.M. Castano, B. Frangione, b-Sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: Implications for Alzheimer’s therapy, Nature Medicine 4 Ž1998. 822–826. E.O. Stejskal, J.E. Tanner, Spin diffusion measurements: spin echoes in the presence of a time-dependent field gradient, J. Chem. Phys. 42 Ž1965. 288–292. E. Terzi, G. Holzemann, J. Seelig, Self-association of beta-amyloid peptide Ž1–40. in solution and binding to lipid membranes, J. Mol. Biol. 252 Ž1995. 633–642. L.O. Tjernberg, J. Naslund, F. Lindqvist, J. Johansson, A.R. ¨ Karlstrom, ¨ J. Thyberg, L. Terenius, C. Nordstedt, Arrest of b-
w46x
w47x
w48x
w49x
w50x
w51x
w52x
w53x
amyloid fibril formation by a pentapeptide ligand, J. Biol. Chem. 271 Ž1996. 8545–8548. L.O. Tjernberg, C. Lilliehook, S. Hahne, ¨¨ J.E. Callaway, J. Naslund, ¨ J. Thyberg, L. Terenius, C. Norstedt, Controlling amyloid b-peptide fibril formation with protease-stable ligands, J. Biol. Chem. 272 Ž1997. 12601–12605. J.O. Tjernberg, J.E. Callaway, A. Tjernberg, S. Hahne, C. Lillehook, ¨¨ L. Terenius, J. Thyberg, C. Nordstedt, A molecular model of Alzheimer’s amyloid b-peptide fibril formation, J. Biol. Chem. 274 Ž1999. 12619–12625. J. Ulas, L.C. Brunner, L. Nguyen, C.W. Cotman, Reduced density of adenosine A1 receptors and preserved coupling of adenosine A1 receptors to G proteins in Alzheimer hippocampus: a quantitative autoradiographic study, Neuroscience 52 Ž1993. 843–854. ¨ Langel, A. Valkna, A. Jureus, ´ E. Karelson, M. Zilmer, T. Bartfai, U. Differential regulation of adenylate cyclase activity in rat ventral and dorsal hippocampus by rat galanin, Neurosci. Lett. 187 Ž1995. 75–78. W. Xia, J. Zhang, D. Kholodenko, M. Citron, M.B. Podlisny, D.B. Teplow, C. Haas, P. Seubert, E.H. Koo, D.J. Selkoe, Enhanced production and oligomerization of the 42-residue amyloid b-protein by chinese hamster ovary cells stably expressing mutant presenilins, J. Biol. Chem. 272 Ž1997. 7977–7982. S.M. Yatin, M. Aksenov, D.A. Butterfield, The antioxidant vitamin E modulates amyloid b-peptide induced creatine kinase activity inhibition and increased protein oxidation: implications for the free radical hypothesis of Alzheimer’s disease, Neurochem. Res. 24 Ž1999. 427–435. S.M. Yatin, M. Yatin, T. Aulick, K.B. Ain, D.A. Butterfield, Alzheimer’s amyloid b-peptide associated free radicals increase rat embryonic neuronal polyamine uptake and ornithine decarboxylase activity: protective effect of vitamin E, Neurosci. Lett. 263 Ž1999. 17–20. ¨ Langel, Differential M. Zorko, M. Pooga, K. Saar, K. Rezaei, U. regulation of GTPase activity by mastoparan and galparan, Arch. Biochem. Biophys. 349 Ž1998. 321–328.