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809 A specific marker of oxidative protein modifications, 2,4-dinitrophenylhydrazine, labels the neurofibrillary pathology of Alzheimer disease
FIFTH INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE course of the disease. This energy failure is assumed to contribute to both abnormal APP processing and hyperphosphorylation of tau protein. The latter abnormalities are crucial events in SDAT.
8O7 EVIDENCE SUPPORTING THE INFLAMMATORY HYPOTHESIS OF ALZHEIMER DISEASE P.L. McGeer*, M. Schulzer and E.G. McGeer Kinsmen Laboratory of Neurological Research, and the Departments of Medicine and Statistics University of British Columbia, 2255 W e s b r o o k Mall, Vancouver, B.C., Canada, V6T 1Z3 A l z h e i m e r d i s e a s e (AD) lesions are c h a r a c t e r i z e d b y the p r e s e n c e of n u m e r o u s i n f l a m m a t o r y proteins. This has led to the h y p o t h e s i s that b r a i n i n f l a m m a t i o n is a cause of neuronal injury in A D a n d that a n t i i n f l a m m a t o r y d r u g s m a y act as protective agents. There are 17 epidemiological studies from nine different countries in w h i c h arthritis, a major indication for the use of a n t i i n f l a m m a t o r y drugs, or a n t i i n f l a m m a t o r y d r u g s themselves, have been considered as risk factors for AD. These studies indicate that both factors are associated w i t h a reduced frequency of AD. The s m a l l size of most s t u d i e s h a s l i m i t e d their i n d i v i d u a l statistical significance, b u t s i m i l a r i t i e s in d e s i g n h a v e m a d e it possible to evaluate c o m b i n e d results. W e u s e d established methods of statistical meta-analysis to e s t i m a t e the overall chance of i n d i v i d u a l s exposed to arthritis or antiinflammat.~ry d r u ~ d e v e l o p i n g AD a~ Lompared to tim general population. Seven case control studies with arthritis as the risk factor y i e l d e d a n overall o d d s ratio of 0.556 (p < 0.0001), w h i l e four case control studies w i t h steroids and three case control studies w i t h nons t e r o i d a l a n t i i n f l a m m a t o r y d r u g s (NSAIDs) yielded odds ratios of 0.656 (p = 0.049) a n d 0.496 (p = 0.0002). When NSAIDs and steroids w e r e c o m b i n e d into a single category of antiinflammatory drugs, the o d d s ratio w a s 0.556 (p < 0.0001). Population based studies were less similar in d e s i g n than case control studies, complicating the process of a p p l y i n g statistical r e c t a - a n a l y t i c a l techniques. Nevertheless, p o p u l a t i o n b a s e d studies w i t h r h e u m a t o i d arthritis and NSAID use as risk factors strongly s u p p o r t e d the results of case control studies. These data s u g g e s t that a n t i i n f l a m m a t o r y d r u g s m a y protect against AD. In one small, double blind clinical trial, progress of the disease was arrested w i t h indomethacin. Further controlled clinical trials are necessary.
8O8 Regulation of Amyloid Precursor Protein Processing and Synthesis by Receptor-mediated Signal Transduetion Robert K. K. Lee*, Wataru Araki and Richard J. Wurtman Department of Brain and Cognitive Sciences, E25-604, Massachusetts Institute of Technology, Cambridge, MA 02139, U.S.A. All peptides which form plaques in Alzheimar's disease (AD) are derived by proteolytic cleavage of a much larger amyloid precursor protein (APP). In cell lines, primary neurons and astrocyte cultures, neurotransmitters that activate cell-surface receptors coupled to phosphotidylinositol (PI) hydrolysis and protein kinase C stimulate secretory cleavage within the A]3 to release the soluble N-terminal ectodomain ofAPP (APPs) into the extracellular space. We recently showed that trans-(lS,3R)-l-amino-l,3-cyclopentane dicarboxylic acid (ACPD, 100gM), acting on metabotropie glutamate receptor (mGluR) subtypes 1 and 5, which are coupled to PI hydrolysis, approximately doubles the amount of APPs secreted by cultured rat hippocampal neurons or by cortical astrocytes, relative to unstimulated control cells. We now report that cAMP-mediated signal transdu~ion regulates both APP processing and synthesis. Increasing intracellular cAMP concentrations in cultured cortical astrocytes or hippocampal neurons, by short-term treatment (15min) with dibutyryl cAMP or forskolin (both 100I~M), potently suppressed the enhanced APPs secretion produced by ACPD (100gM). Preliminary data suggests that this inhibitory effect was mimicked by an agunist of the mGluR subtype 4 (the glutamate #utoreceptor), L(+)-2-amino-4-phosphonobutyrie acid (L-AP4, 1001~M), and by the 13-adrenergic aguhist isoproterenol (1001xM), both of which increased cAMP concentrations in cortical astrocytes. On the other hand, prolonged treatment (24h) of cortical astrocytes with membrane-permeant dibutyryl cAMP, or the l~-edranergic agunists
$201
isoproteranol or L-norepinephdne bitartate (50 or 1001aM), increased the syntheses ofAPP mRNA and protein by -1 fold relative to untreated cells, as measured by Northern and Western blot analyses, These findings show that cAMP interferes with non-amyloidogenie APP processing, and that increases in cAMP may promote AI3 formation through increased APP expression. Thus, alterations in signal transduction events mediated by neurotransmitter receptors may contribute to AD pathophysiology.
809 A Specific Marker of Oxidative Protein Modifications, 2,4-Dinitrophenylhydrazine, Labels the Neurofibriilary Pathology of AIzheimer Disease. Smith M.A.*, Sayre L.M.+, Anderson V.§, Richey P.L., Siedlak S.L., Beal M.F."r, Kowall N.* and Perry G. Institute of Pathology, Departments of +Chemistry and §Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106; tDepartment of Neurology, Harvard Medical School, Department of Neurology, Boston, Massachusetts 02114; +Department of Veterans Affairs, Bedford, Massachusetts 01730. Aggregates of fibrillar insoluble protein, senile plaques and neurofibrillary tangles (NFT), are invariable pathological features of Alzheimer disease (AD). The mechanism of protein aggregation is unknown although recent evidence suggests that amyloid-[3 deposition in senile plaques and x/neurofilament deposition in NFT may involve specific posRranslational modifications mediated by oxidative stress. Oxidative stress affects all of the major cellular macromolecules resulting in, among other things, lipid peroxidation of cellular membranes, DNA modification and protein oxidation. All of these processes result in the formation of macromolecular-based carbonyl groups. In this study, we used 2,4-dinitrophenylhydrazine (DNP), a chemical reagent that reacts with free carbonyls to determine oxidatively-modified molecules in AD. Using DNP, followed by anti-DNP immunocytochemistry, we demonstrated chemical cytoplasm, NFT as well as nuclei of neurons and glia. Age-matched controls showed little to no labeling of these structures. The specificity of the DNP reaction was demonstrated by abolishment of labeling by prior reduction of carbonyls with sodium borohydride (NaBH4). Although the protein component of NFT that contained increased carbonyls was not determined, it is likely that the high lysine content of both "~ and neurofilaments makes these proteins particularly vulnerable to oxidative attack. Interestingly, carbonyls found in nuclei suggests oxidative attack of DNA. Overall, these findings support the notion that oxidative stress plays an important role in the pathogenesis of AD and most importantly, the in rive occurrence of oxidative modifications in NFT suggest that carbonyl-modifieation is associated with a generalized cytoskeletal abnormality that may be critical in the pathogenesis of neurofibrillary pathology. Moreover, extensive oxidative stress-related posttranslational modifications to DNA and lipid membranes might account for the regional neuronal dysfunction and death associated with the pathogenesis of AD. Supported by the American Health Assistance Foundation, the American Federation of Aging Research, and NIH Grants AG09287 and NS22688.
8O7 EVIDENCE SUPPORTING THE INFLAMMATORY HYPOTHESIS OF ALZHEIMER DISEASE P.L. McGeer*, M. Schulzer and E.G. McGeer Kinsmen Laboratory of Neurological Research, and the Departments of Medicine and Statistics University of British Columbia, 2255 W e s b r o o k Mall, Vancouver, B.C., Canada, V6T 1Z3 A l z h e i m e r d i s e a s e (AD) lesions are c h a r a c t e r i z e d b y the p r e s e n c e of n u m e r o u s i n f l a m m a t o r y proteins. This has led to the h y p o t h e s i s that b r a i n i n f l a m m a t i o n is a cause of neuronal injury in A D a n d that a n t i i n f l a m m a t o r y d r u g s m a y act as protective agents. There are 17 epidemiological studies from nine different countries in w h i c h arthritis, a major indication for the use of a n t i i n f l a m m a t o r y drugs, or a n t i i n f l a m m a t o r y d r u g s themselves, have been considered as risk factors for AD. These studies indicate that both factors are associated w i t h a reduced frequency of AD. The s m a l l size of most s t u d i e s h a s l i m i t e d their i n d i v i d u a l statistical significance, b u t s i m i l a r i t i e s in d e s i g n h a v e m a d e it possible to evaluate c o m b i n e d results. W e u s e d established methods of statistical meta-analysis to e s t i m a t e the overall chance of i n d i v i d u a l s exposed to arthritis or antiinflammat.~ry d r u ~ d e v e l o p i n g AD a~ Lompared to tim general population. Seven case control studies with arthritis as the risk factor y i e l d e d a n overall o d d s ratio of 0.556 (p < 0.0001), w h i l e four case control studies w i t h steroids and three case control studies w i t h nons t e r o i d a l a n t i i n f l a m m a t o r y d r u g s (NSAIDs) yielded odds ratios of 0.656 (p = 0.049) a n d 0.496 (p = 0.0002). When NSAIDs and steroids w e r e c o m b i n e d into a single category of antiinflammatory drugs, the o d d s ratio w a s 0.556 (p < 0.0001). Population based studies were less similar in d e s i g n than case control studies, complicating the process of a p p l y i n g statistical r e c t a - a n a l y t i c a l techniques. Nevertheless, p o p u l a t i o n b a s e d studies w i t h r h e u m a t o i d arthritis and NSAID use as risk factors strongly s u p p o r t e d the results of case control studies. These data s u g g e s t that a n t i i n f l a m m a t o r y d r u g s m a y protect against AD. In one small, double blind clinical trial, progress of the disease was arrested w i t h indomethacin. Further controlled clinical trials are necessary.
8O8 Regulation of Amyloid Precursor Protein Processing and Synthesis by Receptor-mediated Signal Transduetion Robert K. K. Lee*, Wataru Araki and Richard J. Wurtman Department of Brain and Cognitive Sciences, E25-604, Massachusetts Institute of Technology, Cambridge, MA 02139, U.S.A. All peptides which form plaques in Alzheimar's disease (AD) are derived by proteolytic cleavage of a much larger amyloid precursor protein (APP). In cell lines, primary neurons and astrocyte cultures, neurotransmitters that activate cell-surface receptors coupled to phosphotidylinositol (PI) hydrolysis and protein kinase C stimulate secretory cleavage within the A]3 to release the soluble N-terminal ectodomain ofAPP (APPs) into the extracellular space. We recently showed that trans-(lS,3R)-l-amino-l,3-cyclopentane dicarboxylic acid (ACPD, 100gM), acting on metabotropie glutamate receptor (mGluR) subtypes 1 and 5, which are coupled to PI hydrolysis, approximately doubles the amount of APPs secreted by cultured rat hippocampal neurons or by cortical astrocytes, relative to unstimulated control cells. We now report that cAMP-mediated signal transdu~ion regulates both APP processing and synthesis. Increasing intracellular cAMP concentrations in cultured cortical astrocytes or hippocampal neurons, by short-term treatment (15min) with dibutyryl cAMP or forskolin (both 100I~M), potently suppressed the enhanced APPs secretion produced by ACPD (100gM). Preliminary data suggests that this inhibitory effect was mimicked by an agunist of the mGluR subtype 4 (the glutamate #utoreceptor), L(+)-2-amino-4-phosphonobutyrie acid (L-AP4, 1001~M), and by the 13-adrenergic aguhist isoproterenol (1001xM), both of which increased cAMP concentrations in cortical astrocytes. On the other hand, prolonged treatment (24h) of cortical astrocytes with membrane-permeant dibutyryl cAMP, or the l~-edranergic agunists
$201
isoproteranol or L-norepinephdne bitartate (50 or 1001aM), increased the syntheses ofAPP mRNA and protein by -1 fold relative to untreated cells, as measured by Northern and Western blot analyses, These findings show that cAMP interferes with non-amyloidogenie APP processing, and that increases in cAMP may promote AI3 formation through increased APP expression. Thus, alterations in signal transduction events mediated by neurotransmitter receptors may contribute to AD pathophysiology.
809 A Specific Marker of Oxidative Protein Modifications, 2,4-Dinitrophenylhydrazine, Labels the Neurofibriilary Pathology of AIzheimer Disease. Smith M.A.*, Sayre L.M.+, Anderson V.§, Richey P.L., Siedlak S.L., Beal M.F."r, Kowall N.* and Perry G. Institute of Pathology, Departments of +Chemistry and §Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106; tDepartment of Neurology, Harvard Medical School, Department of Neurology, Boston, Massachusetts 02114; +Department of Veterans Affairs, Bedford, Massachusetts 01730. Aggregates of fibrillar insoluble protein, senile plaques and neurofibrillary tangles (NFT), are invariable pathological features of Alzheimer disease (AD). The mechanism of protein aggregation is unknown although recent evidence suggests that amyloid-[3 deposition in senile plaques and x/neurofilament deposition in NFT may involve specific posRranslational modifications mediated by oxidative stress. Oxidative stress affects all of the major cellular macromolecules resulting in, among other things, lipid peroxidation of cellular membranes, DNA modification and protein oxidation. All of these processes result in the formation of macromolecular-based carbonyl groups. In this study, we used 2,4-dinitrophenylhydrazine (DNP), a chemical reagent that reacts with free carbonyls to determine oxidatively-modified molecules in AD. Using DNP, followed by anti-DNP immunocytochemistry, we demonstrated chemical cytoplasm, NFT as well as nuclei of neurons and glia. Age-matched controls showed little to no labeling of these structures. The specificity of the DNP reaction was demonstrated by abolishment of labeling by prior reduction of carbonyls with sodium borohydride (NaBH4). Although the protein component of NFT that contained increased carbonyls was not determined, it is likely that the high lysine content of both "~ and neurofilaments makes these proteins particularly vulnerable to oxidative attack. Interestingly, carbonyls found in nuclei suggests oxidative attack of DNA. Overall, these findings support the notion that oxidative stress plays an important role in the pathogenesis of AD and most importantly, the in rive occurrence of oxidative modifications in NFT suggest that carbonyl-modifieation is associated with a generalized cytoskeletal abnormality that may be critical in the pathogenesis of neurofibrillary pathology. Moreover, extensive oxidative stress-related posttranslational modifications to DNA and lipid membranes might account for the regional neuronal dysfunction and death associated with the pathogenesis of AD. Supported by the American Health Assistance Foundation, the American Federation of Aging Research, and NIH Grants AG09287 and NS22688.