- Email: [email protected]
P4-174: Levels of protein carbonyls and nitrotyrosines in cerebrospinal fluid, serum and plasma in Alzheimer's disease
Poster Presentations P4: P4-171
CEREBRAL HYPOPERFUSION-INDUCED OXIDATIVE STRESS AND MITOCHONDRIAL FAILURE AS INITIATORS OF AGING AND ALZHEIMER’S DISEASE PATHOLOGY
Gjumrakch Aliev1, Justin C. Shenk1, Kathryn Fischbach1, Celia J. Cobb1, Gerardo J. Pacheco1, Eldar Gasimov2, George Perry3,4, 1 Department of Biology, and Electron Microscopy Research Center, University of Texas at San Antonio, San Antonio, TX, USA; 2Department of Cytology, Histology and Embryology, Azerbaijan Medical University, Baku, Azerbaijan; 3College of Sciences, University of Texas at San Antonio, San Antonio, TX, USA; 4Case Western Reserve University, Cleveland, OH, USA. Contact e-mail: [email protected] Background: Cerebral hypoperfusion-induced mitochondrial failure appears to be a key pathogenic factor in the development of age-associated diseases, triggering mild cognitive impairment and eventual conversion to Alzheimer’s disease (AD). Mitochondrial integrity is associated with cellular viability. We studied cellular and subcellular features of hippocampal neurons and microvessel mitochondrial lesions, oxidative stress markers and protein immunoreactivity in human AD and animal models that mimic MCI and/or AD. In addition, we studied the effects of dietary antioxidant treatment on neuronal mitochondrial ultrastructure in rats and ApoE4 mice. Methods: In situ hybridization, using mitochondrial DNA (mtDNA) probes for human wild type, 5kb deleted and mouse mtDNA was performed in conjunction with immunocytochemistry using antibodies against APP, 8-hydroxyguanosine, all three isoforms of nitric oxide synthase (neuronal, inducible and endothelial NOS) and cytochrome c oxidase. Mitochondrial degeneration was analyzed under electron microscopy in young and old rats with and without dietary supplementation of mitochondrial antioxidants (lipoic acid and ALCAR) as well as transgenic mice overexpressing either APP or ApoE4. Results: A significantly higher degree of mitochondrial damage was found in neurons and cerebrovascular wall cells in AD and in aged animal models used compared to age-matched controls and non-treated subjects. These abnormalities coexist with overexpression of APP and inducible NOS immunoreactivity and amyloid deposition in the same regions which characterize the presence of large, lipid-laden vacuoles in the cytoplasm of endothelial cells. In addition, in situ hybridization revealed deleted mtDNA positive signals in the damaged mitochondria of neurons, vascular endothelium and perivascular cells. Conclusions: We conclude that blocking the underlying oxidative stress stimuli via treatment with selective pharmacological agents such as selective mitochondrial antioxidants, NO precursors and/or suppressors may normalize the actions of endogenous antioxidant systems and vasoactive substances in brain cells. P4-172
ABETA ACTIVATES CYTOSOLIC PHOSPHOLIPASE A2 THROUGH AN NMDA SIGNALING PATHWAY INVOLVING NADPH OXIDASE IN CORTICAL NEURONS
Grace Y. Sun, Phullara B. Shelat, James C. Lee, Agnes Simonyi, Albert Y. Sun, University of Missouri, Columbia, MO, USA. Contact e-mail: [email protected] Background: Oxidative stress has been implicated in the development of Alzheimer’s disease (AD). Despite yet unknown mechanisms, there is evidence for the involvement of oligomeric Abeta in mediating the oxidative process which in turn, impairs neurons and glial cells. Recent studies in our laboratory demonstrated a novel mechanism linking oligomeric Abeta to production of reactive oxygen species (ROS) through NADPH oxidase. Increase in ROS in astrocytes was shown to activate phospholipases A2 (PLA2) and subsequently alter mitochondrial membrane function (Zhu et al., J. Neurosci. 26: 11111; 2006). PLA2 are ubiquitous enzymes for cleaving fatty acids, particularly arachidonic acid (AA) from membrane phospholipids. Besides serving as a precursor for eicosanoids, AA in neurons may act as a retrograde messenger and has been implicated in modulation of synaptic plasticity and memory functions. In this study,
T721
we investigate whether oligomeric Abeta may alter AA release in neurons and whether this process involves NADPH oxidase and cytosolic PLA2. Methods: Primary cortical neurons were cultured from embryos of 18 day-old pregnant Sprague-Dawley rats. Neurons at 8-10 DIV were treated with oligomeric Abeta42 or NMDA for 30 min and subsequently used for assay of AA release using [14C]-AA, assessment of ROS production using dihydroethidin, and Western blot analysis for phospho-ERK and cPLA2. Results: Both Abeta42 and NMDA stimulated AA release from prelabeled phospholipids in neurons. AA release was attributed to ROS production through NADPH oxidase which in turn, activated ERK1/2 and cPLA2. In addition, Abeta-mediated AA release from neurons was inhibited by AP-5 and memantine, two different NMDA receptor antagonists, suggesting interaction between Abeta and NMDA signaling pathway. Conclusions: This study unveiled a novel mechanism whereby NMDA and oligomeric Abeta, stimulate ROS production through NADPH oxidase and AA release through cPLA2. Activation of cPLA2 also produces lysophospholipids, compounds which can cause perturbation of membranes. These results lend strong support to explaining the oxidative-degradative mechanism of Abeta and signaling pathways leading to impairment of neuronal synaptic plasticity. Understanding this mechanism may enhance the development of novel therapeutic strategies for retarding the progression of AD. P4-173
CYTOPLASMIC GELSOLIN, A BETA-AMYLOID BINDING PROTEIN, IS INDUCED BY OXIDATIVE STRESS IN PC-12 CELLS
Lina Ji, Abha Chauhan, Ved Chauhan, New York State Institute for Basic Research in Developmental Disabilities, New York, NY, USA. Contact e-mail: [email protected] Background: Gelsolin binds to actin and amyloid beta-protein (A), and exists in all cell types including neurons. It plays important roles in cytoskeletal remodeling, apoptosis, and carcinogenesis. Recent evidence from our and other laboratories also suggests a role of gelsolin in regulation of beta-amyloidosis in Alzheimer’s disease (AD). Our previous results showed that both cytoplasmic and plasma gelsolin form a complex with A, irrespective of their differences in amino acid sequences and cysteine thiol groups. Plasma gelsolin inhibited A fibrillization and defibrillized preformed A fibrils. It is known that hydrogen peroxide (H2O2) induces apoptosis in rat pheochromocytoma cell line (PC-12) cells by triggering oxidative stress, and gelsolin has anti-apoptotic property. Oxidative damage and neuronal cell loss are characteristics of AD. Therefore, a relationship between oxidative stress, apoptosis and intracellular gelsolin was envisioned. Methods: Gelsolin, actin, caspase-9 and poly ADP-ribose polymerase (PARP) were probed by western blot. Malonyldialdehyde (MDA), an end product of lipid peroxidation, was measured by thiobarbituric acid. Results: We report that expression of cytoplasmic gelsolin is induced in PC-12 cells by oxidative stress. When PC-12 cells were subjected to oxidative stress, i.e., H2O2, the expression of gelsolin significantly increased in a time- and dose-dependent manner. Meanwhile, cleaved products of procaspase-9 and PARP were observed in the H2O2-treated PC-12 cells. We also found increased MDA, a marker of oxidative stress, in the brains of patients with Alzheimer’s disease as compared to control subjects. Conclusions: These results suggest that oxidative stressor induces expression of gelsolin, which may delay the development of AD by its anti-apoptotic property and interaction with amyloid-beta protein. P4-174
LEVELS OF PROTEIN CARBONYLS AND NITROTYROSINES IN CEREBROSPINAL FLUID, SERUM AND PLASMA IN ALZHEIMER’S DISEASE
Minna A. Korolainen1, Tuula Pirttila¨2, 1University of Kuopio, Kuopio, Finland; 2University of Kuopio and Kuopio University Hospital, Kuopio, Finland. Contact e-mail: [email protected] Background: Alterations in oxidative metabolism have been associated with aging as well as with the pathogenesis of age-related neurodegenera-
T722
Poster Presentations P4:
tive disorders such as Alzheimer’s disease (AD). Carbonyls and nitrotyrosines are considered as markers of oxidative stress on proteins. Several studies have shown differences in carbonylation and nitration of individual proteins in brain and body fluids of AD patients. Methods: We have here assessed total levels of carbonyls and nitrotyrosines in cerebrospinal fluid, serum and plasma from 22 AD patients and 18 age-matched controls using commercially available enzyme immunoassay kits. Results: Decreased protein carbonylation was detected in serum of AD patients as compared to age-matched controls. No differences were observed in the levels of CSF or plasma carbonyls between AD patients and controls. However, decreased CSF protein carbonylation was observed in Apolipoprotein E ⑀4 carriers as compared to non-carriers. Levels of nitrotyrosines did not vary between the groups. Conclusions: According to these data, changes in oxidative metabolism related to the pathogenesis of AD cannot be detected as increased CSF, serum, or plasma protein carbonylation or nitration when compared to age-matched controls. More studies are warranted in order to clarify whether decreased levels of serum carbonyls can be linked to the pathogenesis of AD. P4-175
LOSS OF PHOSPHOLIPID ASYMMETRY AND ELEVATED BRAIN APOPTOTIC PROTEIN LEVELS IN SUBJECTS WITH AMNESTIC MILD COGNITIVE IMPAIRMENT AND ALZHEIMER’S DISEASE
Miranda Lange1, Giovanna Cenini1, Maurizio Memo2, D. Allan Butterfield1, 1University of Kentucky, Lexington, KY, USA; 2 University of Brescia, Brescia, Italy. Contact e-mail: [email protected] Background: Oxidative stress, a hallmark of Alzheimer disease (AD), has been shown to induce lipid peroxidation and apoptosis disrupting cellular homeostasis. Normally, the aminophospholipid phosphatidylserine (PtdSer) is asymmetrically distributed on the cytosolic leaflet of the lipid bilayer. Following lipid peroxidation, asymmetry is altered, characterized by the appearance of PtdSer on the outer leaflet, to initiate the first stages of apoptosis. PtdSer asymmetry is actively maintained by the ATP-dependent aminophospholipid translocase flippase, whose function is inhibited if covalently bound by lipid peroxidation products such as 4-hydroxynonenal (HNE) within the bilayer in which it is produced. Additionally, proapoptotic proteins Bax and caspase-3 have also been implemented in the oxidative modification of PtdSer resulting in subsequent asymmetric collapse, while anti-apoptotic protein Bcl-2 has been found to prevent this process. This investigation focused on the detection of PtdSer outer membrane exposure in synaptosomes from brain of subjects with AD and amnestic mild cognitive impairment (MCI), as well as expression levels of apoptosis-related proteins Bcl-2, Bax, and caspase-3. MCI is considered a possible transition point between normal cognitive aging and probable AD. Brain from subjects with MCI is reported to have increased levels of tissue oxidation; therefore, the results of this study could mark the progression of patients with MCI into AD. Methods: Fluorescence assay of synaptosomes from brain of subjects with AD and MCI were conducted to detect exposed PtdSer, while Western blot together with computer-assisted image analysis was used to quantify expression of pro- and anti- apoptotic proteins Bax, caspase-3, and Bcl-2, respectively. Results: Fluorescence analysis suggest PtdSer exposure is significantly increased in brain from subjects with MCI and AD contributing to the early apoptotic elevation of pro- and antiapoptotic proteins, as detected by Western blot, and ultimately neuronal loss. Conclusions: This study contributes to a model of apoptosis-specific oxidation of PtdSer in AD and MCI brain. These results are consistent with the notion that oxidative stress increases production of lipid peroxidation products and levels of apoptosis-related proteins that affect PtdSer asymmetry, a process required for apoptotic-cell death and clearance. Support: NIH (NIA) grants [AG-05119; AG-10836] to D. A. B.
P4-176
PROTEOMICS DETERMINED GLYCOPROTEOME IN MILD COGNITIVE IMPAIRMENT, EARLY ALZHEIMER’S DISEASE, AND LATE-STAGE ALZHEIMER’S DISEASE HIPPOCAMPUS AND INFERIOR PARIETAL LOBULE
Joshua B. Owen, Fabio Dominico, Marzia Perluigi, Rukhsana Sultana, D. Allan Butterfield, University of Kentucky Department of Chemistry and Sanders-Brown Center on Aging, Lexington, KY, USA. Contact email: [email protected] Background: Post-translational modifications (PTM) have been topics of intense research in the elucidation of Alzheimer’s disease (AD) pathogenesis. Some of these PTMs include carbonylation, nitration, and 4-hydroxynonenalation. Protein glycosylation is a lesser studied PTM, even though it is involved in critical cellular processes including protein folding, protein anchoring to cellular membranes, and protein localization. Little is known about the glycoproteome in mild cognitive impairment (MCI), early Alzheimer’s disease (EAD), and late-stage Alzheimer’s disease (LAD) brain hippocampus and inferior parietal lobule. The current study employed affinity column and proteomics techniques to observe the changes of N-linked glycoproteins in the aforementioned disease brain regions. Methods: Concanavalin A affinity columns were utilized to recognize glycoproteins with mannose and/or terminal glucose moieties. Mannose residue motifs are characteristic of all N-linked glycoproteins. Once the glycoproteins were isolated, 30 KDa concentrators were necessary to obtain protein concentration measurements and for loading purposes. The concentrated aliquots underwent isoelectric focusing, followed by SDSPAGE electrophoresis. PD Quest image analysis was then employed to determine variations in the glycoproteome in MCI, EAD, and LAD hippocampus and inferior parietal lobule brain regions compared to age matched controls. Peptide mass fingerprinting (MALDI-TOF) coupled to data base interrogation identified these variations. Results: Several key proteins, including dihydropyrimidinase related protein-2 (DRP-2), were identified to be differentially expressed in MCI, EAD, and LAD hippocampus and inferior parietal lobule. DRP-2 is involved in neuronal development and dendritic maintenance, as well as dendritic elongation. Others showed dendritic length to be shorter in AD brain. Conclusions: This is the first report of the glycoproteome in MCI, EAD, and LAD hippocampus and inferior parietal lobule. Several key proteins were identified by proteomics analysis as being differential expressed. This differential expression may be indicative of aberrations in glycosylation-mediated processes, such as protein folding and trafficking and cell-cell interactions. Improperly folded and localized proteins would certainly contribute to the pathogenesis of Alzheimer’s disease due to lack of function and improper spatial orientation. This research was supported by NIH grants to D.A.B. P4-177
ASSESSING IMMUNE-RELATED OXIDATIVE STRESS AND PROTEOMICS IN A MOUSE MODEL OF ALZHEIMER’S DISEASE
Rena˜ A. Sowell, D. Allan Butterfield, University of Kentucky, Lexington, KY, USA. Contact e-mail: [email protected] Background: The progressive loss of functional capacity within the immune system, termed “immunosenescence,” is associated with the pathogenesis of age-related diseases including neurodegenerative, cardiovascular, and muscular/skeletal disorders, and leads to a greater susceptibility to infections, tumors and possibly cancer. Recent studies associate immunosenescence with Alzheimer’s disease (AD) and propose that it may be a key contributor to AD pathogenesis. Oxidative stress, which is a pathological hallmark of AD, may also play a role in immunosenescence however, the exact mechanisms governing this phenomenon are not understood. The work presented herein employs a mouse model to investigate the molecular pathways associated with declines in the immune functions in AD. Methods: An initial survey of changes in the following oxidative stress markers has been measured in spleens isolated from AD animals and age-matched controls: protein carbonyls and 3-nitrotyrosine bound pro-
CEREBRAL HYPOPERFUSION-INDUCED OXIDATIVE STRESS AND MITOCHONDRIAL FAILURE AS INITIATORS OF AGING AND ALZHEIMER’S DISEASE PATHOLOGY
Gjumrakch Aliev1, Justin C. Shenk1, Kathryn Fischbach1, Celia J. Cobb1, Gerardo J. Pacheco1, Eldar Gasimov2, George Perry3,4, 1 Department of Biology, and Electron Microscopy Research Center, University of Texas at San Antonio, San Antonio, TX, USA; 2Department of Cytology, Histology and Embryology, Azerbaijan Medical University, Baku, Azerbaijan; 3College of Sciences, University of Texas at San Antonio, San Antonio, TX, USA; 4Case Western Reserve University, Cleveland, OH, USA. Contact e-mail: [email protected] Background: Cerebral hypoperfusion-induced mitochondrial failure appears to be a key pathogenic factor in the development of age-associated diseases, triggering mild cognitive impairment and eventual conversion to Alzheimer’s disease (AD). Mitochondrial integrity is associated with cellular viability. We studied cellular and subcellular features of hippocampal neurons and microvessel mitochondrial lesions, oxidative stress markers and protein immunoreactivity in human AD and animal models that mimic MCI and/or AD. In addition, we studied the effects of dietary antioxidant treatment on neuronal mitochondrial ultrastructure in rats and ApoE4 mice. Methods: In situ hybridization, using mitochondrial DNA (mtDNA) probes for human wild type, 5kb deleted and mouse mtDNA was performed in conjunction with immunocytochemistry using antibodies against APP, 8-hydroxyguanosine, all three isoforms of nitric oxide synthase (neuronal, inducible and endothelial NOS) and cytochrome c oxidase. Mitochondrial degeneration was analyzed under electron microscopy in young and old rats with and without dietary supplementation of mitochondrial antioxidants (lipoic acid and ALCAR) as well as transgenic mice overexpressing either APP or ApoE4. Results: A significantly higher degree of mitochondrial damage was found in neurons and cerebrovascular wall cells in AD and in aged animal models used compared to age-matched controls and non-treated subjects. These abnormalities coexist with overexpression of APP and inducible NOS immunoreactivity and amyloid deposition in the same regions which characterize the presence of large, lipid-laden vacuoles in the cytoplasm of endothelial cells. In addition, in situ hybridization revealed deleted mtDNA positive signals in the damaged mitochondria of neurons, vascular endothelium and perivascular cells. Conclusions: We conclude that blocking the underlying oxidative stress stimuli via treatment with selective pharmacological agents such as selective mitochondrial antioxidants, NO precursors and/or suppressors may normalize the actions of endogenous antioxidant systems and vasoactive substances in brain cells. P4-172
ABETA ACTIVATES CYTOSOLIC PHOSPHOLIPASE A2 THROUGH AN NMDA SIGNALING PATHWAY INVOLVING NADPH OXIDASE IN CORTICAL NEURONS
Grace Y. Sun, Phullara B. Shelat, James C. Lee, Agnes Simonyi, Albert Y. Sun, University of Missouri, Columbia, MO, USA. Contact e-mail: [email protected] Background: Oxidative stress has been implicated in the development of Alzheimer’s disease (AD). Despite yet unknown mechanisms, there is evidence for the involvement of oligomeric Abeta in mediating the oxidative process which in turn, impairs neurons and glial cells. Recent studies in our laboratory demonstrated a novel mechanism linking oligomeric Abeta to production of reactive oxygen species (ROS) through NADPH oxidase. Increase in ROS in astrocytes was shown to activate phospholipases A2 (PLA2) and subsequently alter mitochondrial membrane function (Zhu et al., J. Neurosci. 26: 11111; 2006). PLA2 are ubiquitous enzymes for cleaving fatty acids, particularly arachidonic acid (AA) from membrane phospholipids. Besides serving as a precursor for eicosanoids, AA in neurons may act as a retrograde messenger and has been implicated in modulation of synaptic plasticity and memory functions. In this study,
T721
we investigate whether oligomeric Abeta may alter AA release in neurons and whether this process involves NADPH oxidase and cytosolic PLA2. Methods: Primary cortical neurons were cultured from embryos of 18 day-old pregnant Sprague-Dawley rats. Neurons at 8-10 DIV were treated with oligomeric Abeta42 or NMDA for 30 min and subsequently used for assay of AA release using [14C]-AA, assessment of ROS production using dihydroethidin, and Western blot analysis for phospho-ERK and cPLA2. Results: Both Abeta42 and NMDA stimulated AA release from prelabeled phospholipids in neurons. AA release was attributed to ROS production through NADPH oxidase which in turn, activated ERK1/2 and cPLA2. In addition, Abeta-mediated AA release from neurons was inhibited by AP-5 and memantine, two different NMDA receptor antagonists, suggesting interaction between Abeta and NMDA signaling pathway. Conclusions: This study unveiled a novel mechanism whereby NMDA and oligomeric Abeta, stimulate ROS production through NADPH oxidase and AA release through cPLA2. Activation of cPLA2 also produces lysophospholipids, compounds which can cause perturbation of membranes. These results lend strong support to explaining the oxidative-degradative mechanism of Abeta and signaling pathways leading to impairment of neuronal synaptic plasticity. Understanding this mechanism may enhance the development of novel therapeutic strategies for retarding the progression of AD. P4-173
CYTOPLASMIC GELSOLIN, A BETA-AMYLOID BINDING PROTEIN, IS INDUCED BY OXIDATIVE STRESS IN PC-12 CELLS
Lina Ji, Abha Chauhan, Ved Chauhan, New York State Institute for Basic Research in Developmental Disabilities, New York, NY, USA. Contact e-mail: [email protected] Background: Gelsolin binds to actin and amyloid beta-protein (A), and exists in all cell types including neurons. It plays important roles in cytoskeletal remodeling, apoptosis, and carcinogenesis. Recent evidence from our and other laboratories also suggests a role of gelsolin in regulation of beta-amyloidosis in Alzheimer’s disease (AD). Our previous results showed that both cytoplasmic and plasma gelsolin form a complex with A, irrespective of their differences in amino acid sequences and cysteine thiol groups. Plasma gelsolin inhibited A fibrillization and defibrillized preformed A fibrils. It is known that hydrogen peroxide (H2O2) induces apoptosis in rat pheochromocytoma cell line (PC-12) cells by triggering oxidative stress, and gelsolin has anti-apoptotic property. Oxidative damage and neuronal cell loss are characteristics of AD. Therefore, a relationship between oxidative stress, apoptosis and intracellular gelsolin was envisioned. Methods: Gelsolin, actin, caspase-9 and poly ADP-ribose polymerase (PARP) were probed by western blot. Malonyldialdehyde (MDA), an end product of lipid peroxidation, was measured by thiobarbituric acid. Results: We report that expression of cytoplasmic gelsolin is induced in PC-12 cells by oxidative stress. When PC-12 cells were subjected to oxidative stress, i.e., H2O2, the expression of gelsolin significantly increased in a time- and dose-dependent manner. Meanwhile, cleaved products of procaspase-9 and PARP were observed in the H2O2-treated PC-12 cells. We also found increased MDA, a marker of oxidative stress, in the brains of patients with Alzheimer’s disease as compared to control subjects. Conclusions: These results suggest that oxidative stressor induces expression of gelsolin, which may delay the development of AD by its anti-apoptotic property and interaction with amyloid-beta protein. P4-174
LEVELS OF PROTEIN CARBONYLS AND NITROTYROSINES IN CEREBROSPINAL FLUID, SERUM AND PLASMA IN ALZHEIMER’S DISEASE
Minna A. Korolainen1, Tuula Pirttila¨2, 1University of Kuopio, Kuopio, Finland; 2University of Kuopio and Kuopio University Hospital, Kuopio, Finland. Contact e-mail: [email protected] Background: Alterations in oxidative metabolism have been associated with aging as well as with the pathogenesis of age-related neurodegenera-
T722
Poster Presentations P4:
tive disorders such as Alzheimer’s disease (AD). Carbonyls and nitrotyrosines are considered as markers of oxidative stress on proteins. Several studies have shown differences in carbonylation and nitration of individual proteins in brain and body fluids of AD patients. Methods: We have here assessed total levels of carbonyls and nitrotyrosines in cerebrospinal fluid, serum and plasma from 22 AD patients and 18 age-matched controls using commercially available enzyme immunoassay kits. Results: Decreased protein carbonylation was detected in serum of AD patients as compared to age-matched controls. No differences were observed in the levels of CSF or plasma carbonyls between AD patients and controls. However, decreased CSF protein carbonylation was observed in Apolipoprotein E ⑀4 carriers as compared to non-carriers. Levels of nitrotyrosines did not vary between the groups. Conclusions: According to these data, changes in oxidative metabolism related to the pathogenesis of AD cannot be detected as increased CSF, serum, or plasma protein carbonylation or nitration when compared to age-matched controls. More studies are warranted in order to clarify whether decreased levels of serum carbonyls can be linked to the pathogenesis of AD. P4-175
LOSS OF PHOSPHOLIPID ASYMMETRY AND ELEVATED BRAIN APOPTOTIC PROTEIN LEVELS IN SUBJECTS WITH AMNESTIC MILD COGNITIVE IMPAIRMENT AND ALZHEIMER’S DISEASE
Miranda Lange1, Giovanna Cenini1, Maurizio Memo2, D. Allan Butterfield1, 1University of Kentucky, Lexington, KY, USA; 2 University of Brescia, Brescia, Italy. Contact e-mail: [email protected] Background: Oxidative stress, a hallmark of Alzheimer disease (AD), has been shown to induce lipid peroxidation and apoptosis disrupting cellular homeostasis. Normally, the aminophospholipid phosphatidylserine (PtdSer) is asymmetrically distributed on the cytosolic leaflet of the lipid bilayer. Following lipid peroxidation, asymmetry is altered, characterized by the appearance of PtdSer on the outer leaflet, to initiate the first stages of apoptosis. PtdSer asymmetry is actively maintained by the ATP-dependent aminophospholipid translocase flippase, whose function is inhibited if covalently bound by lipid peroxidation products such as 4-hydroxynonenal (HNE) within the bilayer in which it is produced. Additionally, proapoptotic proteins Bax and caspase-3 have also been implemented in the oxidative modification of PtdSer resulting in subsequent asymmetric collapse, while anti-apoptotic protein Bcl-2 has been found to prevent this process. This investigation focused on the detection of PtdSer outer membrane exposure in synaptosomes from brain of subjects with AD and amnestic mild cognitive impairment (MCI), as well as expression levels of apoptosis-related proteins Bcl-2, Bax, and caspase-3. MCI is considered a possible transition point between normal cognitive aging and probable AD. Brain from subjects with MCI is reported to have increased levels of tissue oxidation; therefore, the results of this study could mark the progression of patients with MCI into AD. Methods: Fluorescence assay of synaptosomes from brain of subjects with AD and MCI were conducted to detect exposed PtdSer, while Western blot together with computer-assisted image analysis was used to quantify expression of pro- and anti- apoptotic proteins Bax, caspase-3, and Bcl-2, respectively. Results: Fluorescence analysis suggest PtdSer exposure is significantly increased in brain from subjects with MCI and AD contributing to the early apoptotic elevation of pro- and antiapoptotic proteins, as detected by Western blot, and ultimately neuronal loss. Conclusions: This study contributes to a model of apoptosis-specific oxidation of PtdSer in AD and MCI brain. These results are consistent with the notion that oxidative stress increases production of lipid peroxidation products and levels of apoptosis-related proteins that affect PtdSer asymmetry, a process required for apoptotic-cell death and clearance. Support: NIH (NIA) grants [AG-05119; AG-10836] to D. A. B.
P4-176
PROTEOMICS DETERMINED GLYCOPROTEOME IN MILD COGNITIVE IMPAIRMENT, EARLY ALZHEIMER’S DISEASE, AND LATE-STAGE ALZHEIMER’S DISEASE HIPPOCAMPUS AND INFERIOR PARIETAL LOBULE
Joshua B. Owen, Fabio Dominico, Marzia Perluigi, Rukhsana Sultana, D. Allan Butterfield, University of Kentucky Department of Chemistry and Sanders-Brown Center on Aging, Lexington, KY, USA. Contact email: [email protected] Background: Post-translational modifications (PTM) have been topics of intense research in the elucidation of Alzheimer’s disease (AD) pathogenesis. Some of these PTMs include carbonylation, nitration, and 4-hydroxynonenalation. Protein glycosylation is a lesser studied PTM, even though it is involved in critical cellular processes including protein folding, protein anchoring to cellular membranes, and protein localization. Little is known about the glycoproteome in mild cognitive impairment (MCI), early Alzheimer’s disease (EAD), and late-stage Alzheimer’s disease (LAD) brain hippocampus and inferior parietal lobule. The current study employed affinity column and proteomics techniques to observe the changes of N-linked glycoproteins in the aforementioned disease brain regions. Methods: Concanavalin A affinity columns were utilized to recognize glycoproteins with mannose and/or terminal glucose moieties. Mannose residue motifs are characteristic of all N-linked glycoproteins. Once the glycoproteins were isolated, 30 KDa concentrators were necessary to obtain protein concentration measurements and for loading purposes. The concentrated aliquots underwent isoelectric focusing, followed by SDSPAGE electrophoresis. PD Quest image analysis was then employed to determine variations in the glycoproteome in MCI, EAD, and LAD hippocampus and inferior parietal lobule brain regions compared to age matched controls. Peptide mass fingerprinting (MALDI-TOF) coupled to data base interrogation identified these variations. Results: Several key proteins, including dihydropyrimidinase related protein-2 (DRP-2), were identified to be differentially expressed in MCI, EAD, and LAD hippocampus and inferior parietal lobule. DRP-2 is involved in neuronal development and dendritic maintenance, as well as dendritic elongation. Others showed dendritic length to be shorter in AD brain. Conclusions: This is the first report of the glycoproteome in MCI, EAD, and LAD hippocampus and inferior parietal lobule. Several key proteins were identified by proteomics analysis as being differential expressed. This differential expression may be indicative of aberrations in glycosylation-mediated processes, such as protein folding and trafficking and cell-cell interactions. Improperly folded and localized proteins would certainly contribute to the pathogenesis of Alzheimer’s disease due to lack of function and improper spatial orientation. This research was supported by NIH grants to D.A.B. P4-177
ASSESSING IMMUNE-RELATED OXIDATIVE STRESS AND PROTEOMICS IN A MOUSE MODEL OF ALZHEIMER’S DISEASE
Rena˜ A. Sowell, D. Allan Butterfield, University of Kentucky, Lexington, KY, USA. Contact e-mail: [email protected] Background: The progressive loss of functional capacity within the immune system, termed “immunosenescence,” is associated with the pathogenesis of age-related diseases including neurodegenerative, cardiovascular, and muscular/skeletal disorders, and leads to a greater susceptibility to infections, tumors and possibly cancer. Recent studies associate immunosenescence with Alzheimer’s disease (AD) and propose that it may be a key contributor to AD pathogenesis. Oxidative stress, which is a pathological hallmark of AD, may also play a role in immunosenescence however, the exact mechanisms governing this phenomenon are not understood. The work presented herein employs a mouse model to investigate the molecular pathways associated with declines in the immune functions in AD. Methods: An initial survey of changes in the following oxidative stress markers has been measured in spleens isolated from AD animals and age-matched controls: protein carbonyls and 3-nitrotyrosine bound pro-