- Email: [email protected]
Phospholipase pathway in Alzheimer's disease brains: decrease in Gαi in dorsolateral prefrontal cortex
Molecular Brain Research 66 Ž1999. 188–190
Short communication
Phospholipase pathway in Alzheimer’s disease brains: decrease in Ga i in dorsolateral prefrontal cortex Robert A. Young, Konrad Talbot, Zhi-yong Gao, John Q. Trojanowski, Bryan A. Wolf
)
Department of Pathology and Laboratory Medicine, UniÕersity of PennsylÕania School of Medicine, 230 John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104-6082, USA Accepted 29 December 1998
Abstract There is substantial evidence that G-protein-associated signaling pathways in the brain are altered in Alzheimer’s disease ŽAD.. Using quantitative immunoblotting we find a significant decrease in Ga i levels in every AD case examined compared to controls Žmean Ga i level in AD was 43.5 " 7.4% of control.. Ga o levels were slightly decreased, but Ga q and bg were normal. Phospholipase C-b1, but not g1, levels were also decreased. Total phospholipase C activity and ceramide levels were not changed. Thus, in AD, there is impairment in the Ga i-associated signaling pathway in neurons. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Phospholipase C; Alzheimer’s disease; G-protein
There has been increasing evidence suggesting that the phospholipase C signal transduction pathway is abnormal in Alzheimer’s disease ŽAD. w2,6,13,18x. Muscarinic–Gprotein receptor coupling and select G-protein levels are reportedly normal or decreased w9–11,13,14,17x. The purpose of this study was to examine the protein levels of G-proteins and phospholipase C isoforms in AD frontal cortex. Postmortem tissue were obtained from AD w7x patients and controls without evidence of AD coming to autopsy at the Hospital of the University of Pennsylvania. Patients were age-matched, had a similar sex distribution, similar post-mortem interval, and similar brain weights ŽTable 1.. Tissue blocks from the dorsolateral prefrontal cortex Žwithin Brodmann areas 9, 10 and 46. were collected at autopsy, immediately frozen and stored at y808C. An olive-size piece of tissue Žgrey matter. was removed from each human postmortem dorsolateral prefrontal cortex sample Ž200–500 mg wet tissue weight. and homogenized in 10 mM HEPES pH 7.4, 1 mM EGTA, 1 mM MgCl 2 , supplemented with 115 mM PMSF, 1 mM pepstatin, and 21 mM leupeptin. Two to twenty-five micrograms of protein in 20 ml of loading buffer Ž70 mM Tris pH 6.7, 16
) Corresponding author. [email protected]
Fax:
q 1-215-573-2266;
E-mail:
M urea, 6.0% SDS, 100 mM dithiothreitol, 0.005% Bromophenol blue. were analyzed by immunoblotting as described w8x. The following antisera were used for Western blotting: anti-Ga i 8730 Žimmunogen ŽKLH.KNNLKDCGLF., anti-Ga o 9072 Žimmunogen ŽKLH.ANNLRGCGLY., anti-Ga q 946 Žimmunogen ŽKLH.QLNLKEYNLV., anti-Gb g 5357 Žbovine brain bg .. Antibodies to phospholipase C isoforms were obtained from Upstate Biotechnology ŽLake Placid, NY, USA.. Phospholipase C assay was performed as reported w1x, while enzymatic measurement of ceramide was a modification of a previously described assay for diacylglycerol mass w16x. There was a dramatic decrease in Ga i levels in every AD case examined compared to controls ŽFig. 1.. Mean Ga i level was 43.5 " 7.4% of control Ž P - 0.0001, n s 14, Fig. 2.. Phospholipase C-b1 levels was lower in AD frontal cortex Ž53.1 " 10.5% of control n s 13, P s 0.02.. In contrast, phospholipase C-g1 levels, total phospholipase C activity and ceramide levels were not changed in AD compared to control Ždata not shown.. Previous studies of G-protein levels in AD brains have essentially reported normal levels of most G-proteins w10– 13,15,17x. An earlier study reported that Ga i levels in frontal cortex, hippocampus and cerebellum were normal in AD w11x. In one study, however, a very slightly decreased level of Ga i in the superior temporal gyrus was reported w10x. Another study reported a nearly 60% reduc-
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 0 2 3 - 6
R.A. Young et al.r Molecular Brain Research 66 (1999) 188–190
189
Table 1 Summary of case data
Number of cases Age Žmean"S.E.M.. Sex Žfemalermale. Post-mortem interval Žmean"S.E.M., h. Brain weight Žmean"S.E.M., g. Duration of dementia Žmean"S.E.M., years.
Normal
AD
14 69.5"5.5 9r5 10.0"1.4 1189"45 –
15 77.3"2.1 11r4 10.6"1.2 1137"20 8.6"1.2
tion in Ga i in the temporal cortex of AD post-mortem brain, and a 37% reduction in the angular gyrus w12x. This last study is consistent with the substantial decrease in Ga i levels found in the frontal cortex reported herein. There are three subtypes of Ga i , Ga i1 , Ga i2 and Ga i3 . The antibody used in the present studies is more selective for Ga i1 and Ga i2 but also cross-reacts with Ga i3 w3x. Agonist-stimulated w35 SxGTPgS binding to Gi and Go was decreased in AD, suggesting that there may be a decoupling between receptors and associated G proteins w17x. Since Ga i inhibits adenylate cyclase, a decrease in Ga i levels could result in altered G-protein inhibition of adenylate cyclase. However, a number of studies have convincingly shown that adenylate cyclase activity as well as associated receptors coupled in an inhibitory fashion are not affected by AD w5x. A more likely effector coupled to Ga i that may be affected in AD
Fig. 2. Selective decrease in Ga i and Ga o but not Ga q and bg-subunit protein levels in frontal cortex of AD patients. Frontal cortex from normal controls and AD patients was homogenized, analyzed by 10% SDS-PAGE, immunoblotted with an antibody to Ga o wa9072x, Ga q wa946x, Ga i wa8730x, and bg-subunit wa5357x and quantitated with a PhosphorImager using w125 Ixprotein-A. Results are shown as the protein levels of the various G-protein a-subunits and bg-subunit expressed as the % Žmean" S.E.M.. of the control levels from 8 to 14 observations per condition. Normal controls: hatched bars; AD patients: solid bars. ) P - 0.05 vs. control; )) P - 0.0001 vs. control.
is phospholipase A 2 . There is increasing evidence that certain isoforms of phospholipase A 2 are coupled to Ga i w4x. It is conceivable that the decrease in Ga i1 levels in AD brain results in decreased signaling through the phospholipase A 2 pathway.
Acknowledgements This study was supported by the National Institute of Aging grants AG09215 and AG11542, the Penn Alzheimer’s Disease Core Center Pilot Grant Program NIA AG10124, the Hartford Foundation Program for Research on Aging, and the William Pepper Fund of the University of Pennsylvania. B.A.W. is a recipient of NIH Research Career Development Award K04 DK02217. We are grateful to Chris Major for assistance in developing the ceramide assay.
References
Fig. 1. Decreased Ga i protein level in frontal cortex of AD patients. Top panel: Immunoblot of Ga i protein level in frontal cortex of eight controls and eight AD patients. An equal amount of protein Ž25 mg. was loaded in each lane. Bottom panel: Quantitation of the data from the top panel expressed in PhosphorImager arbitrary units. Normal controls, hatched bars wCx; Alzheimer’s disease patients, solid bars wADx.
w1x C.A. Alter, M. Amagasu, K. Shah, Y.C. Jolly, C. Major, B.A. Wolf, U-73122 does not specifically inhibit phospholipase C in rat pancreatic islets and insulin-secreting beta-cell lines, Life Sci. 54 Ž1994. PL107–PL112. w2x J. Bothmer, J. Jolles, Phosphoinositide metabolism, aging and Alzheimer’s disease, Biochim. Biophys. Acta Mol. Basis Dis. 1225 Ž1994. 111–124. w3x L.F. Brass, D.R. Manning, A.G. Williams, M.J. Woolkalis, M. Poncz, Receptor and G protein-mediated responses to thrombin in HEL cells, J. Biol. Chem. 266 Ž1991. 958–965. w4x J.R. Burke, L.B. Davern, K.R. Gregor, G. Todderud, J.G. Alford, K.M. Tramposch, Phosphorylation and calcium influx are not sufficient for the activation of cytosolic phospholipase A2 in U937 cells: requirement for a Gi alpha-type G-protein, Biochim. Biophys. Acta 1341 Ž1997. 223–237.
190
R.A. Young et al.r Molecular Brain Research 66 (1999) 188–190
w5x C.J. Fowler, A. Garlind, C. O’Neill, R.F. Cowburn, Receptor–effector coupling dysfunctions in Alzheimer’s disease, Ann. NY Acad. Sci. 786 Ž1996. 294–304. w6x C. Jolly-Tornetta, Z. Gao, V.M.Y. Lee, B.A. Wolf, Regulation of amyloid precursor protein secretion by glutamate receptors in human ntera 2 neurons win process citationx, J. Biol. Chem. 273 Ž1998. 14015–14021. w7x Z.S. Khachaturian, Diagnosis of Alzheimer’s disease, Arch. Neurol. 42 Ž1985. 1097–1105. w8x R.J. Konrad, R.A. Young, R.D. Record, R.M. Smith, P. Butkerait, D. Manning, L. Jarett, B.A. Wolf, The heterotrimeric G-protein Gi is localized to the insulin secretory granules of b-cells and is involved in insulin exocytosis, J. Biol. Chem. 270 Ž1995. 12869– 12876. w9x C.J. Ladner, G.G. Celesia, D.J. Magnuson, J.M. Lee, Regional alterations in M 1 muscarinic receptor–G protein coupling in Alzheimer’s disease, J. Neuropathol. Exp. Neurol. 54 Ž1995. 783– 789. w10x X.H. Li, A.F. Greenwood, R. Powers, R.S. Jope, Effects of postmortem interval, age, and Alzheimer’s disease on G-proteins in human brain, Neurobiol. Aging 17 Ž1996. 115–122. w11x M. McLaughlin, B.M. Ross, G. Milligan, J. McCulloch, J.T. Knowler, Robustness of G proteins in Alzheimer’s disease: an immunoblot study, J. Neurochem. 57 Ž1991. 9–14.
w12x C. O’Neill, B. Wiehager, C.J. Fowler, R. Ravid, B. Winblad, R.F. Cowburn, Regionally selective alterations in G protein subunit levels in the Alzheimer’s disease brain, Brain Res. 636 Ž1994. 193–201. w13x M.A. Pacheco, R.S. Jope, Phosphoinositide signaling in human brain. wReviewx w65 refs.x, Prog. Neurobiol. 50 Ž1996. 255–273. w14x B.D. Pearce, L.T. Potter, Coupling of m1 muscarinic receptors to G protein in Alzheimer disease, Alzheimer Dis. Assoc. Disord. 5 Ž1991. 163–172. w15x 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. w16x P.P. Van Veldhoven, W.R. Bishop, D.A. Yurivich, R.M. Bell, Ceramide quantitation: evaluation of a mixed micellar assay using E. coli diacylglycerol kinase, Biochem. Mol. Biol. Int. 36 Ž1995. 21–30. w17x H.-Y. Wang, E. Friedman, Receptor-mediated activation of G proteins is reduced in postmortem brains from Alzheimer’s disease patients, Neurosci. Lett. 173 Ž1994. 37–39. w18x B.A. Wolf, A.M. Wertkin, Y.C. Jolly, R.P. Yasuda, B.B. Wolfe, R.J. Konrad, D. Manning, S. Ravi, J.R. Williamson, V.M.Y. Lee, Muscarinic regulation of Alzheimer’s disease amyloid precursor protein secretion and amyloid b-protein production in human neuronal NT2N cells, J. Biol. Chem. 270 Ž1995. 4916–4922.
Short communication
Phospholipase pathway in Alzheimer’s disease brains: decrease in Ga i in dorsolateral prefrontal cortex Robert A. Young, Konrad Talbot, Zhi-yong Gao, John Q. Trojanowski, Bryan A. Wolf
)
Department of Pathology and Laboratory Medicine, UniÕersity of PennsylÕania School of Medicine, 230 John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104-6082, USA Accepted 29 December 1998
Abstract There is substantial evidence that G-protein-associated signaling pathways in the brain are altered in Alzheimer’s disease ŽAD.. Using quantitative immunoblotting we find a significant decrease in Ga i levels in every AD case examined compared to controls Žmean Ga i level in AD was 43.5 " 7.4% of control.. Ga o levels were slightly decreased, but Ga q and bg were normal. Phospholipase C-b1, but not g1, levels were also decreased. Total phospholipase C activity and ceramide levels were not changed. Thus, in AD, there is impairment in the Ga i-associated signaling pathway in neurons. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Phospholipase C; Alzheimer’s disease; G-protein
There has been increasing evidence suggesting that the phospholipase C signal transduction pathway is abnormal in Alzheimer’s disease ŽAD. w2,6,13,18x. Muscarinic–Gprotein receptor coupling and select G-protein levels are reportedly normal or decreased w9–11,13,14,17x. The purpose of this study was to examine the protein levels of G-proteins and phospholipase C isoforms in AD frontal cortex. Postmortem tissue were obtained from AD w7x patients and controls without evidence of AD coming to autopsy at the Hospital of the University of Pennsylvania. Patients were age-matched, had a similar sex distribution, similar post-mortem interval, and similar brain weights ŽTable 1.. Tissue blocks from the dorsolateral prefrontal cortex Žwithin Brodmann areas 9, 10 and 46. were collected at autopsy, immediately frozen and stored at y808C. An olive-size piece of tissue Žgrey matter. was removed from each human postmortem dorsolateral prefrontal cortex sample Ž200–500 mg wet tissue weight. and homogenized in 10 mM HEPES pH 7.4, 1 mM EGTA, 1 mM MgCl 2 , supplemented with 115 mM PMSF, 1 mM pepstatin, and 21 mM leupeptin. Two to twenty-five micrograms of protein in 20 ml of loading buffer Ž70 mM Tris pH 6.7, 16
) Corresponding author. [email protected]
Fax:
q 1-215-573-2266;
E-mail:
M urea, 6.0% SDS, 100 mM dithiothreitol, 0.005% Bromophenol blue. were analyzed by immunoblotting as described w8x. The following antisera were used for Western blotting: anti-Ga i 8730 Žimmunogen ŽKLH.KNNLKDCGLF., anti-Ga o 9072 Žimmunogen ŽKLH.ANNLRGCGLY., anti-Ga q 946 Žimmunogen ŽKLH.QLNLKEYNLV., anti-Gb g 5357 Žbovine brain bg .. Antibodies to phospholipase C isoforms were obtained from Upstate Biotechnology ŽLake Placid, NY, USA.. Phospholipase C assay was performed as reported w1x, while enzymatic measurement of ceramide was a modification of a previously described assay for diacylglycerol mass w16x. There was a dramatic decrease in Ga i levels in every AD case examined compared to controls ŽFig. 1.. Mean Ga i level was 43.5 " 7.4% of control Ž P - 0.0001, n s 14, Fig. 2.. Phospholipase C-b1 levels was lower in AD frontal cortex Ž53.1 " 10.5% of control n s 13, P s 0.02.. In contrast, phospholipase C-g1 levels, total phospholipase C activity and ceramide levels were not changed in AD compared to control Ždata not shown.. Previous studies of G-protein levels in AD brains have essentially reported normal levels of most G-proteins w10– 13,15,17x. An earlier study reported that Ga i levels in frontal cortex, hippocampus and cerebellum were normal in AD w11x. In one study, however, a very slightly decreased level of Ga i in the superior temporal gyrus was reported w10x. Another study reported a nearly 60% reduc-
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 0 2 3 - 6
R.A. Young et al.r Molecular Brain Research 66 (1999) 188–190
189
Table 1 Summary of case data
Number of cases Age Žmean"S.E.M.. Sex Žfemalermale. Post-mortem interval Žmean"S.E.M., h. Brain weight Žmean"S.E.M., g. Duration of dementia Žmean"S.E.M., years.
Normal
AD
14 69.5"5.5 9r5 10.0"1.4 1189"45 –
15 77.3"2.1 11r4 10.6"1.2 1137"20 8.6"1.2
tion in Ga i in the temporal cortex of AD post-mortem brain, and a 37% reduction in the angular gyrus w12x. This last study is consistent with the substantial decrease in Ga i levels found in the frontal cortex reported herein. There are three subtypes of Ga i , Ga i1 , Ga i2 and Ga i3 . The antibody used in the present studies is more selective for Ga i1 and Ga i2 but also cross-reacts with Ga i3 w3x. Agonist-stimulated w35 SxGTPgS binding to Gi and Go was decreased in AD, suggesting that there may be a decoupling between receptors and associated G proteins w17x. Since Ga i inhibits adenylate cyclase, a decrease in Ga i levels could result in altered G-protein inhibition of adenylate cyclase. However, a number of studies have convincingly shown that adenylate cyclase activity as well as associated receptors coupled in an inhibitory fashion are not affected by AD w5x. A more likely effector coupled to Ga i that may be affected in AD
Fig. 2. Selective decrease in Ga i and Ga o but not Ga q and bg-subunit protein levels in frontal cortex of AD patients. Frontal cortex from normal controls and AD patients was homogenized, analyzed by 10% SDS-PAGE, immunoblotted with an antibody to Ga o wa9072x, Ga q wa946x, Ga i wa8730x, and bg-subunit wa5357x and quantitated with a PhosphorImager using w125 Ixprotein-A. Results are shown as the protein levels of the various G-protein a-subunits and bg-subunit expressed as the % Žmean" S.E.M.. of the control levels from 8 to 14 observations per condition. Normal controls: hatched bars; AD patients: solid bars. ) P - 0.05 vs. control; )) P - 0.0001 vs. control.
is phospholipase A 2 . There is increasing evidence that certain isoforms of phospholipase A 2 are coupled to Ga i w4x. It is conceivable that the decrease in Ga i1 levels in AD brain results in decreased signaling through the phospholipase A 2 pathway.
Acknowledgements This study was supported by the National Institute of Aging grants AG09215 and AG11542, the Penn Alzheimer’s Disease Core Center Pilot Grant Program NIA AG10124, the Hartford Foundation Program for Research on Aging, and the William Pepper Fund of the University of Pennsylvania. B.A.W. is a recipient of NIH Research Career Development Award K04 DK02217. We are grateful to Chris Major for assistance in developing the ceramide assay.
References
Fig. 1. Decreased Ga i protein level in frontal cortex of AD patients. Top panel: Immunoblot of Ga i protein level in frontal cortex of eight controls and eight AD patients. An equal amount of protein Ž25 mg. was loaded in each lane. Bottom panel: Quantitation of the data from the top panel expressed in PhosphorImager arbitrary units. Normal controls, hatched bars wCx; Alzheimer’s disease patients, solid bars wADx.
w1x C.A. Alter, M. Amagasu, K. Shah, Y.C. Jolly, C. Major, B.A. Wolf, U-73122 does not specifically inhibit phospholipase C in rat pancreatic islets and insulin-secreting beta-cell lines, Life Sci. 54 Ž1994. PL107–PL112. w2x J. Bothmer, J. Jolles, Phosphoinositide metabolism, aging and Alzheimer’s disease, Biochim. Biophys. Acta Mol. Basis Dis. 1225 Ž1994. 111–124. w3x L.F. Brass, D.R. Manning, A.G. Williams, M.J. Woolkalis, M. Poncz, Receptor and G protein-mediated responses to thrombin in HEL cells, J. Biol. Chem. 266 Ž1991. 958–965. w4x J.R. Burke, L.B. Davern, K.R. Gregor, G. Todderud, J.G. Alford, K.M. Tramposch, Phosphorylation and calcium influx are not sufficient for the activation of cytosolic phospholipase A2 in U937 cells: requirement for a Gi alpha-type G-protein, Biochim. Biophys. Acta 1341 Ž1997. 223–237.
190
R.A. Young et al.r Molecular Brain Research 66 (1999) 188–190
w5x C.J. Fowler, A. Garlind, C. O’Neill, R.F. Cowburn, Receptor–effector coupling dysfunctions in Alzheimer’s disease, Ann. NY Acad. Sci. 786 Ž1996. 294–304. w6x C. Jolly-Tornetta, Z. Gao, V.M.Y. Lee, B.A. Wolf, Regulation of amyloid precursor protein secretion by glutamate receptors in human ntera 2 neurons win process citationx, J. Biol. Chem. 273 Ž1998. 14015–14021. w7x Z.S. Khachaturian, Diagnosis of Alzheimer’s disease, Arch. Neurol. 42 Ž1985. 1097–1105. w8x R.J. Konrad, R.A. Young, R.D. Record, R.M. Smith, P. Butkerait, D. Manning, L. Jarett, B.A. Wolf, The heterotrimeric G-protein Gi is localized to the insulin secretory granules of b-cells and is involved in insulin exocytosis, J. Biol. Chem. 270 Ž1995. 12869– 12876. w9x C.J. Ladner, G.G. Celesia, D.J. Magnuson, J.M. Lee, Regional alterations in M 1 muscarinic receptor–G protein coupling in Alzheimer’s disease, J. Neuropathol. Exp. Neurol. 54 Ž1995. 783– 789. w10x X.H. Li, A.F. Greenwood, R. Powers, R.S. Jope, Effects of postmortem interval, age, and Alzheimer’s disease on G-proteins in human brain, Neurobiol. Aging 17 Ž1996. 115–122. w11x M. McLaughlin, B.M. Ross, G. Milligan, J. McCulloch, J.T. Knowler, Robustness of G proteins in Alzheimer’s disease: an immunoblot study, J. Neurochem. 57 Ž1991. 9–14.
w12x C. O’Neill, B. Wiehager, C.J. Fowler, R. Ravid, B. Winblad, R.F. Cowburn, Regionally selective alterations in G protein subunit levels in the Alzheimer’s disease brain, Brain Res. 636 Ž1994. 193–201. w13x M.A. Pacheco, R.S. Jope, Phosphoinositide signaling in human brain. wReviewx w65 refs.x, Prog. Neurobiol. 50 Ž1996. 255–273. w14x B.D. Pearce, L.T. Potter, Coupling of m1 muscarinic receptors to G protein in Alzheimer disease, Alzheimer Dis. Assoc. Disord. 5 Ž1991. 163–172. w15x 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. w16x P.P. Van Veldhoven, W.R. Bishop, D.A. Yurivich, R.M. Bell, Ceramide quantitation: evaluation of a mixed micellar assay using E. coli diacylglycerol kinase, Biochem. Mol. Biol. Int. 36 Ž1995. 21–30. w17x H.-Y. Wang, E. Friedman, Receptor-mediated activation of G proteins is reduced in postmortem brains from Alzheimer’s disease patients, Neurosci. Lett. 173 Ž1994. 37–39. w18x B.A. Wolf, A.M. Wertkin, Y.C. Jolly, R.P. Yasuda, B.B. Wolfe, R.J. Konrad, D. Manning, S. Ravi, J.R. Williamson, V.M.Y. Lee, Muscarinic regulation of Alzheimer’s disease amyloid precursor protein secretion and amyloid b-protein production in human neuronal NT2N cells, J. Biol. Chem. 270 Ž1995. 4916–4922.