Over four million individuals in the United States currently suffer from Alzheimer\u27s disease (AD), a devastating disorder of progressive dementia. Within the next several decades, AD is expected to affect over 22 million people globally. AD can only be definitively diagnosed by postmortem examination. Thus, investigation into the specific pathogenesis of neuronal degeneration and death in AD on a biochemical level is essential for both earlier diagnosis and potential treatment and prevention options. Overproduction of amyloid [3-peptide (A[3) in the brain leads to both free radical oxidative stress and toxicity to neurons in AD. My undergraduate biochemical studies with regard to AD explore the various ways in which free radical oxidative stress might contribute to the pathology of AD. In particular, this review highlights studies using Af3-precursor mutations in animal models, and analysis of histone-DNA interactions

Petroze, Robin

English

University of Kentucky

KaleidoscopeVolume 2 Article 152003The Role of Oxidative Stress in Alzheimer's DiseaseRobin PetrozeUniversity of KentuckyFollow this and additional works at: https://uknowledge.uky.edu/kaleidoscopePart of the Medical Biochemistry CommonsRight click to open a feedback form in a new tab to let us know how this document benefitsyou.This Beckman Scholars Program is brought to you for free and open access by the The Office of Undergraduate Research at UKnowledge. It has beenaccepted for inclusion in Kaleidoscope by an authorized editor of UKnowledge. For more information, please contact UKnowledge@lsv.uky.edu.Recommended CitationPetroze, Robin (2003) "The Role of Oxidative Stress in Alzheimer's Disease," Kaleidoscope: Vol. 2, Article 15.Available at: https://uknowledge.uky.edu/kaleidoscope/vol2/iss1/1560 BECKMAN ScHOLARS PROGRAM                                              KALEIDOSCOPE FALL 2 0 0 3 Abstract Over four million individuals in the United States currently suffer from Alzheimer's disease (AD), a devastating disorder of progressive dementia. Within the next several decades, AD is expected to affect over 22 million people globally. AD can only be definitively diagnosed by postmortem exami-nation. Thus, investigation into the specific patho-genesis of neuronal degeneration and death in AD on a biochemical level is essential for both earlier diagnosis and potential treatment and prevention options. Overproduction of amyloid [3-peptide (A[3) in the brain leads to both free radical oxidative stress and toxicity to neurons in AD. My under-graduate biochemical studies with regard to AD explore the various ways in which free radical oxi-dative stress might contribute to the pathology of AD. In particular, this review highlights studies using Af3-precursor mutations in animal models, and analysis of histone-DNA interactions. Introduction Acknowledged as a disease of progressive demen-tia as early as 1910 by German psychologist Alois Alzheimer, questions concerning the specifics of Alzheimer's disease (AD) still plague the scientific community. With the modern burgeoning of tech-nological approaches in molecular biology and bio-chemistry, recent research has opened the door to understanding the basic mechanisms of the disor-der (Markesbery, 1997). Over four million indi-viduals in the United States currently suffer from AD, the fourth leading cause of death in the United States. Within the next several decades, AD is ex-pected to affect over 22 million people globally (Butterfield et al., 2001}. Characterized by the progressive loss of memory and cognition, major pathological l BECK-;_,~N SCHOLAR -- - -- ::__ _ hallmarks of the disease include neurofibrillary tangles (NFTs) , senile (neuritic) plaques (SPs), and synapse loss (Katzman and Saitoh, 1991) . Amyloid 13-peptide (AI3), in the form of insoluble fibril deposits, is the primary component of senile plaques. Currently, AD can only be definitively diagnosed by postmortem examination (Varadarajan et al., 2000) . Thus , investigation into the specific pathogenesis of neuronal degeneration and death in AD on a biochemical level is essential for earlier diagnosis and potential treatment and prevention options. Known risk factors for AD include: age, family history of dementia or AD, low educational level, low linguistic ability during early stages, and the presence of apolipoprotien E (APO-E) alleles. Of the APO-E2, APO-E3 , and APO-E4 alleles present in humans, the APO-E4 allele signifies a greater incidence of AD (Markesbery, 1997) . My undergraduate biochemical research with re-gard to AD explores the various ways in which free radical oxidative stress might contribute to the pa-thology of AD. Oxidative stress occurs when there are more reactive species (radicals) than antioxidant defenses and repair capacities in a system, such as the brain. In particular, this review will highlight stud-ies using Al3-precursor mutations in animal models and analysis of histone-DNA interactions in AD. Role of A(3 in Oxidative Stress Our laboratory has proposed that overproduction of Al3leads to both free radical oxidative stress and tox-icity to neurons in AD (Butterfield et al ., 2001 ; 2002; Butterfield and Lauderback, 2002; Varadarajan et al., 2000). By inserting itself into neuronal membranes, soluble, aggregated Al3 may induce the formation of reactive oxygen species and reactive nitrogen species that lead to lipid peroxidation, protein oxidation, and protein modification by 4-hydroxy-2-trans-nonenal (HNE) and acrolein, the reactive products of lipid peroxidation. Furthermore, Al3-associated free-radi-cal oxidative stress can affect free fatty-acid release (leading to tau polymerization), disruption of Ca2 + homeostasis, peroxynitrite formation, inflammatory response, impairment of mitochondria, and apoptosis. Al3-associated oxidation may also play a role in the modification and inhibition of several neuronal and glial transmembrane transport systems. This includes ion-motive ATPases, glutamate transporters, glucose transporters, GTP-coupled transmembrane signaling proteins, and polyamine transporters; functional loss in these systems proves destructive to the neurons, causing cell potential loss, excitotoxic glutamate ac-cumulation, decreased glucose availability, decreased intracellular communication, and increased neurotox-ROBIN PETROZE icity (Butterfield et al., 2001 ; 2002; Butterfield and Lauderback, 2002) . The amyloid 13-peptide is derived from the normal processing of the amyloid precursor protein (APP). As Al3 is a normal soluble component found in plasma and cerebrospinal fluid, it is suspected that conver-sion of soluble Al3 into insoluble fibrils may signify a critical step in disease onset, a sign of overproduction of Al3 (Varadarajan et al. , 2000). Presenilin-1 (PS1) appears to cause cleavage of APP, leading to increased Al3 production (Selkoe, 2001). Fibrillar Al3 binding to receptors increases advanced glycation end-products to induce oxidative stress and activate microglia (Yan et al., 1996) . Neurodegenerative sites in the AD brain corre-spond to the presence of oxidative stress and increased Al3 deposits (Hensley et al., 1995) . Given the broad scope of cellular modifications observed in AD and the increased Al3 deposits in AD pathology, a free radi-cal oxidation cascade, in which structural and func-tional alterations would be seen with any protein or lipid structure attacked by a free radical, is feasible as a model for neuronal death in AD (Varadarajan et al., 2000). Genetic Mutations: Studies of Amyloid Precursor Protein and Presenilin Genes Studies in familial AD indicate the central role of Al3 in AD pathogenesis and the presence of Al3-precursor mutations; specifically, mutations in the amyloid pre-cursor protein (APP) and presenilin genes lead to over-production of Al3 and to AD. As in Down's trisomy, APP is expressed on Chromosome 21; consequently, Down's syndrome patients exhibit increased Al3 de-posits and eventually develop AD (Varadarajan et al., 2000) . PS1 cleavage of APP could stimulate faulty processing of Al3 and increased Al3 production (Selkoe, 2001). While the number of sporadic cases of AD over-shadows the occurrence of familial AD cases, a com-mon pathogenesis leading to senile plaque and neurofibrillary tangle formation is postulated for all forms of AD, corresponding to the similar neuropatho-logic lesions seen in both familial and sporadic cases. Therefore, research in familial AD using transgenic mouse models is applicable to all forms of the disease (Kurt et al., 2001). Mice overexpressing mutant human APP genes in a single transgenic model show evidence of develop-ing fibrillar cerebral Ab deposits similar to the Al3 de-posits seen in AD. Cerebral Al3 deposits increase as well in single transgenic mouse models overexpressing mutant or wild-type human PS1 proteins. Yet, the single transgenic models do not show increased Al3 deposits 62 BECKMAN SCHOLARS PROGRAM until later in adulthood. In APP /PS1 double mutant models, the mice develop increased fibrillar A[3 de-posits in the hippocampus and cerebral cortex earlier than single transgenic mice (Kurt et al., 2001). Methods Related to Markers of Oxidative Stress Oxidative stress measures for protein oxidation and lipid peroxidation can be performed on synaptosomes prepared from brain samples obtained from the mu-tant mice. Protein oxidation is measured through the ultra-violet (UV)-Vis, fluorescence, or immunochemi-cal detection of protein carbonyl formation. Free radi-cal attack on susceptible amino acid side chains can induce protein oxidation to form protein carbonyls, as can the interaction of proteins with the products of glycation and glycoxidation, or the products of lipid peroxidation such as acrolein and 4-hydroxy-2-transnonenal (HNE). Protein carbonyls then react with 2,4-dinitrophenylhydrazine to form a protein-bound hydrazone. This hydrazone is then detected using spectroscopic or immunochemical methods (Butterfield et al. , 2001). Due to the unsaturated bonds in the fatty acid b-chain and the high solubility of nonpolar, paramag-netic oxygen in lipid bilayers, like those found in membranes, oxidative stress is especially dangerous for lipids. Initiated by a free radical attack on a lipid, peroxyl radicals are formed when the lipid radical re-acts with oxygen. These peroxyl radicals can react with other lipids to sustain the chain reaction. Many of the products of lipid peroxidation, such as the un-saturated aldehydes acrolein and HNE, are toxic and act to induce oxidative modification of nearby pro-teins. This chain reaction leads to dysfunctional pro-teins and neuronal death (Butterfield et al., 2002) . A crude assessment of lipid peroxidation is the measurement of thiobarbituric acid reactive substances (TBARS) . Because thiobarbituric acid is highly reac-tive with non-lipid moieties as well as lipid peroxidation products, TBARS provide a non-specific marker of lipid peroxidation in AD brains. TBARS fol-low a thiobarbituric acid reaction and can be used to measure molondialdehyde, a secondary product of lipid peroxidation, through fluorescence studies (Butterfield et al ., 2001) . Because HNE is a reactive product of lipid peroxidation, HNE adduct formation measures also provide a marker of lipid peroxidation. HNE adduct formation is quantified using an immunoblotting technique to signify protein modifi-cation as a secondary product of lipid peroxidation (Butterfield et al ., 2002) . KALEIDOSCOPE FALL 2 0 0 3 Role of Antioxidants This research will lead to studies to determine if the administration of various antioxidants, such as N-acetyl cysteine (NAC) has an effect in slowing the manifestation of degenerative symptoms in oxidative stress conditions. NAC upregulates glutathione syn-thesis, providing the limiting amino acid cysteine. Glu-tathione is present in millimolar levels, is the most abundant endogenous antioxidant in the brain, and may act as the first line of defense against oxidative stress. Our laboratory has previously shown that intraparitoneal (i.p.) injection of NAC in mice pro-tects against in-vitro hydroxyl radical-, acrolein-, and peroxynitrite-induced damage (Pocernich et al. , 2000; 2001; Koppal et al., 1999). Furthermore, our laboratory's studies indicate that i.p. injection of NAC protects against the Complex 2 inhibitor, 3-Nitropionic acid, an agent that leads to pathology reminiscent of Huntington's disease (La Fontaine et al., 2000). From the mice that have been fed/injected with NAC, fu-ture studies will measure protein carbonyl formation, TBARS, and HNE to determine the relative effect of an antioxidant in slowing the neurodegeneration in mutant mice. Antioxidants such as Vitamin E (a-, b-, g-, d-toco-pherols and tocotrienols) also exhibit potential for treat-ment or prevention options. Vitamin E is the major chain-breaking antioxidant that protects against lipid peroxidation at the early stages of free radical attack. The a-tocopherol form has been shown to reduce neu-rotoxicity of A[3 in neuronal cell culture (Yatin et al., 2000). The antioxidant role of a-tocopherol is most supported in AD, and it may play a role in the inhibi-tion of glutamate-induced cell death (Halliwell, 2001). DNA Oxidation and Histone Studies In compacting into chromatin, DNA winds around an octamer of core histones. These histones, H1, H2A, H2B, H3, and H4, are rich in lysines in the N-termi-nus, allowing for close interaction with DNA and ad-jacent nucleosomes. Lysine is a basic amino acid, which allows for a strong ionic interaction with DNA (Butterfield and Lauderback, 2002). Histone acetyla-tion, the substitution of an acetyl group for a hydro-gen atom, weakens these interactions, allowing for the chromatin to open up to transcriptional factors (Li etal. , 1993). An imbalance in pro-oxidant over anti-oxidant factors characterizes an oxidatively stressed system, such as a brain in AD. As noted above, free radicals can cause a cascade of oxidative stress that includes protein oxidation and lipid peroxidation, but DNA f ( BECKMAN ScHOLA-=-..:..:R ______ _ oxidation also plays a role in the cascade of oxidative stress in AD. The DNA oxidation marker 8-hydroxy-2-deoxyguanosine has been shown to be increased in mitochondrial and nuclear DNA in AD brain (Mecocci et al., 1994). His tones, the DNA binding proteins, are also susceptible to oxidative stress, as has been shown occurring with hydrogen peroxide treatment (Ullrich et al., 1999). HNE, a reactive end product of lipid peroxidation, can bind to lysine residues by Michael addition to cause protein modification; Michael addition is an organic reaction in which a ketone or an aldehyde is converted to an enolate or an imine in the presence of a base (Esterbauer et al., 1991). HNE significantly alters the conformation of cortical synaptosomal mem-brane proteins, causing dysfunctional proteins (Subramaniam et al. , 1997}. Studies have shown that HNE is increased in AD brains (Markesbery and Lovell, 1998) and AD ventricular fluid (Lovell et al., 1997}. HNE binds to and likely explains the loss of function of the major glutamate transporter in an AD brain (Lauderback et al., 2001) . Because it is present in the AD system and because it causes dysfunctional pro-teins through oxidative modification, HNE may play a role in DNA oxidation in AD. Our laboratory has hypothesized that HNE causes conformational changes to histones, thus altering their DNA binding capabilities. DNA damage in AD may result subsequent to the disruption of histone-DNA interactions. Furthermore, HNE binding to histones may block histone acetylation, thereby potentially al-tering both histone-DNA interactions and transcrip-tion factor penetration. These hypotheses are tested in various ways. Electron paramagnetic resonance (EPR) spin-labeling shows whether or not HNE binds to histones and changes their conformation. Disturb-ing the ionic strength should lessen the ionic histone-DNA interaction; thus, differing the physiological ionic conditions may show an effect of HNE on histone-DNA interaction. Gel electrophoresis is also used to analyze the effect of HNE on histone acetylation. A manuscript describing the results of this study is nearly ready for submission for peer-review (Drake et al. , 2003). Conclusions Current research indicates that Alzheimer's disease (AD) arises due to neurodegeneration promoted by oxidative stress in the brain and the overproduction of amyloid ~-peptide (A~) . This oxidative stress can take the form of protein oxidation, lipid peroxidation, or DNA oxidation, which confound normal brain pro-ROBIN PETROZE cesses by disrupting lipid structure, protein function, and DNA binding capabilities. Prevention hope lies in the use of antioxidants such as Vitamin E and pub-lic acknowledgment of the risk factors such as low education and linguistic capacities. Antioxidants like N-acetyl cysteine (NAC) also pose a future option for treatment. Because of the increasing life span, the effects of aging and neurodegeneration are becoming more prominent in the population, with the potential to evolve into a public health crisis in the near future. Considerable research currently focuses on possible treatment pathways and preventive measures for neurodegenerative diseases like AD. For the Bibliography, see the on-line version of this article at www.uky.edu/kaleidoscope/fall2003. ( THE UNIVERSITY OF KENTUCKY JOURNAL OF UNDERGRADUATE SCHOLARSHI P: 63 

The Role of Oxidative Stress in Alzheimer\u27s Disease

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The Role of Oxidative Stress in Alzheimer\u27s Disease

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