Neurochemical Research, Vol. 24, No. 6, 1999, pp. 767-774

The Expression of Several Mitochondrial and NuclearGenes Encoding the Subunits of Electron TransportChain Enzyme Complexes, Cytochrome c Oxidase,and NADH Dehydrogenase, in Different BrainRegions in Alzheimer's Disease

Michael Y. Aksenov,1,6 H. Michael Tucker,1,2 Prakash Nair,1,2 Marina V. Aksenova,3

D. Allan Butterfield,1,4 Steven Estus,1,2 and William R. Markesbery1,5

(Accepted January 21, 1999)

In this study, changes of the expression of two mitochondrial and two nuclear genes encodingthe subunits of cytochrome c oxidase (CO) and NADH dehydrogenase (ND) were studied in thehippocampus, inferior parietal lobule, and cerebellum of 10 Alzheimer's disease (AD) and 10age-matched control subjects. The altered proportion between CO II and CO IV mRNAs wasobserved in the AD brain. Changes of the proportion between CO II and CO IV transcripts maycontribute to the kinetic perturbation of CO documented in AD. A coordinated decrease of ND4and ND15 mRNAs was found in the AD hippocampus and inferior parietal lobule, but not incerebellum. The decrease of ND4 gene expression may lead to the inhibition of normalubiquinone oxidoreductase activity of ND. This study suggests that changes of the expressionof mitochondrial and nuclear genes, encoding parts of ND and CO enzyme complexes, may con-tribute to alterations of oxidative metabolism in AD.

KEY WORDS: Alzheimer's disease; gene expression; cytochrome c oxidase: NADH dehydrogenase;mRNA; RT PCR.

INTRODUCTION

Alzheimer's disease (AD) is hypothesized to beassociated with oxidative and bioenergetic stress(1-4). Defective mitochondrial electron transport sys-tems have been suggested to be one of the specificsources of oxidative stress in AD (5-9). Studies of1 Sanders-Brown Center on Aging2 Department of Physiology3 Department of Pharmacology4 Department of Chemistry and Center of Membrane Sciences5 Departments of Pathology and Neurology, University of Kentucky,

Lexington, KY.6 Address reprint requests to: Dr. Michael Y. Aksenov, 101 Sanders-

Brown Bldg., University of Kentucky, Lexington, KY 40536. Tel:(606)257-2862 Fax: (606)323-1981 E-mail: mikealO.uky.cam-pus.mci.net.

7670364-3190/99/0600-0767$16.00/0 © 1999 Plenum Publishing Corporation

electron transport chain (ETC) enzyme activitiesdemonstrated that ETC is defective in AD brain, andthe defect centers about cytochrome c oxidase (CO)(5,6,10). A generalized depression of all ETC com-plexes occurs in AD brain mitochondria (5), and thisfinding is supported by in vivo imaging using positronemission tomography, which demonstrates a reductionof oxidative glucose metabolism and ATP productionin the brain of AD patients (11-14). The decrease ofCO activity is the most consistent change of ETC en-zyme activity in AD. Deficits of other ETC enzyme ac-

Abbreviations used: AD (Alzheimer's disease); ETC (electrontransport chain); CO (cytochrome c oxidase); ND (NADH dehydro-genase); HIPP (hippocampus); IPL (inferior parietal lobule); CER(cerebellum)

768 Aksenov et al.

tivities found in several AD brain regions are lesspronounced (5,6).

Defects in the expression of mitochondrial sub-units of NADH dehydrogenase enzyme complex (ND)in the AD brain were reported recently (15,16). Daveyet al. (17) reported that ND activity has a major con-trol of oxidative phosphorylation in mitochondria,such that if a threshold of 25% inhibition of complexI activity is exceeded energy metabolism is severelyimpaired. Thus, even a moderate decrease of ND ac-tivity can make a significant contribution to the devel-opment of bioenergetic stress in AD.

Although a decrease of CO activity in AD hasbeen documented in numerous studies (10,18-21),the molecular basis of CO defect is not understood.The results of immunochemical analyses of CO lev-els in AD are controversial. Wong-Riley et al. (8)recently demonstrated a statistically significant re-duction of CO protein content in the AD visual cor-tex. However, Schagger and Ohm reported that theCO enzyme complex is present in normal concentra-tions in the AD brain (22). Two studies demonstratedmRNAs, encoding different CO subunits are de-creased in brain regions affected by AD pathology(16,23).

CO, purified from the AD brain, displayed anom-alous kinetic behavior compared with control CO (24).Changes of the kinetic properties of CO in AD may re-sult from mutations in genes encoding CO subunits(25), from posttranslational oxidative damage of CO(26), or from the selective decrease in the productionof mRNAs for CO subunits that form the catalytic do-main of the enzyme complex (23). However, the exactmechanism of the kinetic perturbation and decrease ofcomplex IV activity in AD remains unclear.

The enzyme complexes, which catalyze electrontransfer, proton translocation and oxidative phospho-rylation in eukaryotic cells, consist of a large numberof polypeptide subunits encoded by genes from the mi-tochondrial and nuclear genomes. CO, the terminalETC enzyme complex, is composed of 9 (yeast) to 13(bovine) subunits in a 1: 1 stoichiometry (27). The ac-tive center of the enzyme is formed by the three mito-chondrial- encoded subunits (CO I-III), while the re-maining six to ten subunits are nuclear-encoded (28).The mitochondrial ND enzyme complex from bovineheart mitochondria contains about 40 different poly-peptides. Seven of them are encoded in the mitochon-drial DNA (29). The expression of ETC enzyme sub-units encoded by mitochondrial and nuclear DNA isregulated proportionately in different cell and tissuetypes, including the CNS (30,31). Thus, the assembly

and function of ETC enzyme complexes is dependenton coordinated expression of the genes located in twodifferent informational systems of the cell.

Evidence indicates that alterations of gene expres-sion occur in the AD brain (32). Neuronal and glialcells in the AD neocortex display an increased chro-matin condensation (33), which is known to be associ-ated with a reduced level of transcription (34). Ox-idative modification of DNA in AD (35-37) mayaffect the level and accuracy of the transcription ofnuclear and mitochondrial genes. It is possible thatAD pathology differentially affects nuclear and mito-chondrial gene expression, and, therefore, may dys-regulate the production of some ETC enzyme subunitsencoded by mitochondrial or nuclear genomes. Thisstudy determined the ratios of mitochondrial- andnuclear- encoded mRNAs for the subunits of CO andND enzyme complexes in different regions of the ADand control brain.

EXPERIMENTAL PROCEDURE

Tissue Use. Brain specimens for this study were obtained at au-topsy from 10 AD patients (mean age 77 ± 1.2 years) and 10 age-matched control subjects (mean age 86 ± 2.3 years). Mean post-mortem interval was 3.0 ± 0.2 hours for AD patients and 3.2 ±0.3 hours for control subjects. All AD patients had the clinical di-agnoses of probable AD using NINCDS-ADRDA Work Group Cri-teria (38). Histopathological examination of sections from multipleneocortical areas, hippocampus, amygdala and other subcortical,brainstem and cerebellar areas using hematoxylin and eosin andthe modified Bielschowsky stains along with 10D-5 (for amyloidB-peptide) and ubiquitin immunochemistry, revealed that all ADpatients met accepted standard criteria for the histopathological diag-nosis of AD (39-41).

Controls were individuals without a history of dementia, otherneurological diseases, or systemic diseases affecting the brain. Allcontrol subjects were from the University of Kentucky normal vol-unteer control group who underwent annual neuropsychologicaltesting. Neuropathological evaluation of control brains revealed nosignificant gross alterations and only age-associated microscopicchanges.

Brain specimens for the study were removed rapidly at autopsy,immediately placed in liquid nitrogen, and stored at —70°C.

RNA Isolation, Reverse Transcription, and PCR Amplification.Total RNA was extracted from frozen brain samples (100-200 mgtissue) following the method of Chromzynski and Sacchi (42). RNAconcentration was determined by 260/280 absorbance measurement.The integrity of the extracted RNAs was monitored using 1%agarose gel electrophoresis (43).

The RT PCR-based method used in this study was previouslyvalidated as a tool for the analysis of the expression of specificmRNAs (44). For reverse transcription, 1 ug of total RNA was mixedwith 500 pmol of random hexamers (Boehringer Mannheim, Indi-anapolis, IN) in a volume of 20ul, incubated at 95°C for 2 min, andthen placed on ice. A stock solution was then added such that thefinal reaction volume of 30 u1 contained 200 U of Superscript,

Expression of mRNAs for Cytochrome c Oxidase and NADH Dehydrogenase in Alzheimer's Disease

500 uM dNTPs, 40 U of RNAsin, IX reaction buffer (Life Tech-nologies, Rockville, MD). The solution was incubated at 20°C for10 min and at 42°C for 50 min, and the Superscript reverse tran-scriptase was inactivated by heating to 95°C for 2 min. The Super-script was omitted from some samples used as a control for possibleDNA contamination in PCR amplification ("No RT" controls).

Stock PCR reaction mixtures were prepared on ice and con-tained 100 uM each of dATP, dTTP, dGTP, and dCTP, 1.5 mMMgCl2, 1 X reaction buffer, 0.03 U/ul of Taq DNA Polymerase (LifeTechnologies, Rockville, MD). The stock solutions were separatedinto 14.6 ul aliquots, and 0.4 ul of cDNA synthesized in the reversetranscription was added to each aliquot. The reaction mixtures werecovered with a drop of mineral oil and subjected to various cyclesof PCR. The use of multiple cycles allowed us to determine the min-imum number of cycles necessary to detect PCR product and therebystay in the linear region of PCR. Typical reaction conditions were1 min at 94°C, 1 min at 55°C, and 2 min at 72°C. After amplifica-tion cDNAs were separated by polyacrylamide electrophoresis. Gelswere stained with SYBR Green I (Molecular Probes, Inc., Eugene,OR), scanned, and digitized using the FUJIFILM Fluorescent ImageAnalyzer (FUJI, Japan).

Primer Design. Sense and antisense primers for CO subunit II(mitochondrial-encoded) and subunit IV (nuclear-encoded), and forND subunit 4 (mitochondrial-encoded) and subunit 15 (nuclear-encoded) were designed using OLIGO software (National Bio-Sciences, Plymouth, MN). BLAST 2.0 (45) program was used toperform the Internet-based (National Center for BiotechnologyWEB site) homology search for each pair of primers. The pair ofprimers was considered to be acceptable if no significant homol-ogy (less than 50%) with known human sequences other than tar-get was found.

The CO subunit II amplification fragment was derived fromH.sapiens CO II mRNA sequence (emb/X55654) and comprised a123-bp fragment positioned at 7806-7928 bp in human mitochondr-ial genome cDNA sequence (numbers are given according to Ander-son and co-authors (46)). The sequences of the primers and optimizednumber of PCR cycles for CO II gene expression analysis were:

sense primer 5'-TCCTCATCGCCCTCCCATCCC-3'antisense primer 5'-CGCCGTTAGTCGGTGTACTCGT-3'Number of PCR cycles: 15The CO subunit IV amplification fragment was derived from

H.sapiens CO IV mRNA sequence (emb/X54802) and comprised a183-bp fragment (position 355-538 according to/X54802 CO IVmRNA sequence). The sequences of the primers and optimized num-ber of PCR cycles for CO IV gene expression analysis were:

sense primer 5'-ACGAGTGGAAGACGGTTGTGG-3'antisense primer 5'-TGGAGGCTAAGCCCTGGATGG-3'Number of PCR cycles: 22The ND subunit 4 amplification fragment was derived from H.

sapiens human mitochondrial genome cDNA sequence (gb/J01415)and comprised a 373-bp fragment (position 11656-12028 in humanmitochondrial genome cDNA sequence as reported). The sequencesof the primers and optimized number of PCR cycles for ND4 gene ex-pression analysis were:

sense primer 5'-AGCCATTCTCATCCAAACCCC-3'antisense primer 5'-AATGTGGTGGGTGAGTGAGCC-3'Number of PCR cycles: 15The ND subunit 15 amplification fragment was derived from

H.sapiens ND15 mRNA sequence and comprised a 281-bp fragment(sequence code: gb/AF04734, position 42-322). The sequences ofthe primers and optimized number of PCR cycles for ND15 gene ex-pression analysis were:

sense primer 5'-AAGAGTCAAGGGCACGAGCAT-3'antisense primer 5'-TCCCGCTGCTTCCTGATGGTA-3'Number of PCR cycles: 24.Statistical Analysis. Statistical comparisons were made using

ANOVA followed by Dunnett's test for multiple comparisons.

RESULTS

The data described below represent the results ofimage analysis of specific PCR products separated bypolyacrylamide gel electrophoresis. All PCR mixtureswere shown to contain a single amplification product ofthe designed size (Fig. 1) and the identity of these prod-ucts was confirmed by sequencing. To determine if thegene expression of mitochondrial-encoded and nuclear-encoded subunits of CO and ND is discoordinated inAD brain, the CO II/CO IV and ND4/ND15 mRNA ra-tios were quantified, averaged and compared in the hip-pocampus, inferior parietal lobule, and cerebellum ofeach AD patient and control subject studied.

CO H, CO IV Gene Expression and Changes ofCO II/CO IV mRNA Ratio in Three Brain Regions inAD. Representative gel images, which illustrate the re-sults of RT PCR analysis of the expression of CO IIand CO IV genes, are shown in Figure 2A.

The level of CO II message was moderately de-creased in the AD hippocampus and cerebellum (TableIA, P < 0.05). The level of CO II-specific amplifica-tion product in the AD hippocampus was 23% lower

Fig. 1. CO and ND-specific RT PCR products separated by poly-acrylamide electrophoresis. One ug of total human brain RNA wasreverse transcribed with random hexamers and then equal volume(0.4 ul) of total cDNA obtained was amplified with a pair of primersspecific for CO II, CO IV, ND4, or ND15. Reaction mixtures weresubjected to optimized number of PCR cycles.

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770 Aksenov et al.

Fig. 2. Representative gel images illustrating the results of RT PCR analysis of mRNA levels for mitochondrial and nuclear-encoded subunitsof cytochrome oxidase (CO) (Panel A) and NADH dehydrogenase (ND) (Panel B) in three different brain regions of control and AD subjects.Yields of specific RT PCR products were determined using fluorescent image analyzer and used to quantify CO II/CO IV and ND4/ND15mRNA ratios in the hippocampus, inferior parietal lobule, and cerebellum of each control and AD subject.

than in control. In the AD cerebellum the level of COH-specific cDNA fragment was decreased by 19% com-pared to controls.

An overall decrease of CO IV mRNA contentwas observed in all AD brain regions studied (TableIB, P < 0.05). In the AD hippocampus, inferior parietallobule and cerebellum the yield of CO IV-specificcDNA fragment was decreased by 38-40% comparedto controls.

A statistically significant (P < 0.05) 43-51% in-crease in CO II/CO IV mRNA ratio was observed inall AD brain regions studied (Fig. 3A).

ND4, ND15 Gene Expression and Changes ofND4/N15 mRNA Ratio in Three Brain Regions in AD.Fig. 2B shows representative results of the RT PCRanalysis of ND 4 and ND 15 gene expression in threedifferent regions of the control and AD brain. De-creased mean levels of mitochondrial-encoded ND4mRNA were found in the hippocampus and inferiorparietal lobule in AD (Table IC, P < 0.05). The meanlevel of mtDNA-encoded ND4 mRNA in the ADhippocampus and inferior parietal lobule was 50% and

35% lower than in control subjects. The mean level ofnuclear-encoded ND15 mRNA in the hippocampusand inferior parietal lobule of AD subjects was de-creased by 33% and 36% (Table ID, P < 0.05). No sig-nificant changes of the mean level of ND4 or ND15mRNA were observed in the AD cerebellum. TheND4/ND15 mRNA ratios did not change significantly(Fig. 3B) in hippocampus, inferior parietal lobule, andcerebellum of AD subjects.

DISCUSSION

In this study, we demonstrated that changes in thecontent of the message for mitochondrial- encoded andnuclear-encoded subunits of CO and ND ETC com-plexes occur in three different regions of the AD brain.Our findings of moderately decreased mRNA levelsfor CO II in the AD hippocampus are consistent withthe results of in situ hybridization analysis reportedpreviously by Simonian and co-authors (23). In con-trast to the data of these authors, who did not observe

a significant change of CO IV mRNA content in theAD hippocampus, we found that the level of CO IVtranscript is decreased in hippocampus, inferior pari-etal lobule, and cerebellum of AD subjects. Our resultsare consistent with the data of Chandrasekaran andco-authors (16), who reported a decrease of CO IVmRNA in the midtemporal cortex in AD.

According to our results, an alteration of the pro-portion between CO II and CO IV mRNAs occurs in theAD brain. Mitochondrial CO I, CO II and CO III genesencode polypeptides, which form the active site of COETC complex. The CO IV polypeptide, encoded by nu-clear gene in mammalian cells, plays a major role in as-sembly of CO enzyme complex and redox-linked pro-ton translocation (28,47,48). The decrease of thecontent of CO I and CO III mRNAs in the AD brain wasreported previously (11). The results of the subsequentstudy of the CO IV mRNA level in AD led the authorsto the conclusion that coordinated reduction of mito-chondrial and nuclear CO transcripts is responsible forthe decrease of CO activity in the AD brain (16). It hasto be noted, however, that coordinated decrease of themitochondrial- and nuclear-encoded CO mRNAs wouldexplain the deficit of CO activity, but would not explain

the altered kinetic parameters of the enzyme complexpurified from the AD brain (24). A recent study byDavis et al. (25) suggested that specific missense muta-tions in CO I and CO II genes account for the defect ofCO function in AD. However, this finding was criti-cized by Hirano and co-authors (49), who reported thatthe apparent mtDNA heteroplasmy in AD patients andin normal controls was due to the coamplification of au-thentic mtDNA-encoded CO genes together with CO-like mtDNA pseudogenes embedded in nuclear DNA.

Changes of the proportion between the levels ofCO II and CO IV transcripts in the AD brain, observedin our study, suggest that the expression of severalmitochondrial and nuclear genes encoding parts of theactive center of CO may be differentially affected inAD. If the change of the CO II/CO IV mRNA ratiocauses the subsequent change of the CO II/CO IV pro-tein ratio, the assembly and function of the enzymecomplex may be disturbed, which, in turn, may explainaltered kinetic properties of CO in AD, observed byParker and Parks (24).

Levels of both ND4 and ND15 RNAs were de-creased in hippocampus and inferior parietal lobule, butnot in the cerebellum of AD subjects. Our finding of the

Expression of mRNAs for Cytochrome c Oxidase and NADH Dehydrogenase in Alzheimer's Disease 771

Table I. The Expression of mRNAs for Mitochondrial-Encoded and Nuclear-EncodedSubunits of Cytochrome c Oxidase and NADH Dehydrogenase in Different Regions

of AD and Control Brain

A. COII mRNAHIPPIPLCER

B. COIV mRNAHIPPIPLCER

C. ND4 mRNAHIPPIPLCER

D. ND15 mRNAHIPPIPLCER

Control

12122+/-44113167+/-96311335+/-658

12660+/-109011937+/-134714271+/-988

12962+/-171111364+/-14409693+/-1776

12353+/-117215637+/-163211665+/-978

AD

937S+/-49511766+/-6819198+/-495

7331+/-10497151+/5998599+/-900

6469+/-677346+7-799

10463+/-1579

8213+/-13359968+7-7729543+/-1127

% of Contr.

778981

586060

5065

108

676482

P value

0.00062NS

0.018

0.00240.00450.00049

0.0020.027

NS

0.00760.0057

NS

The expression of CO II, CO IV, ND4, and ND15 mRNAs was determined in the hippocampus(HIPP), inferior parietal lobule (IPL), and cerebellum (CER) of AD (n = 10) and age-matchedcontrol (n = 10) subjects.The results of image analysis of specific RT PCR product yields are presented as mean arbitraryintensity units+/-SEM.NS - The difference between AD and Controls was not significant at 95% level (P > 0.05).

772 Aksenov et al.

Fig. 3. CO II/CO IV (A) and ND4/ND 15 (B) mRNA ratios and inthe hippocampus (HIPP), inferior parietal lobule (IPL), andcerebellum (CER) of AD and control subjects. The results arepresented as mean+/-SEM. n = 10, *- P < 0.05.

decreased level of ND4 mRNA is consistent with theresults of other authors, who reported the decreaseof mitochondrial-encoded ND4 (15) and ND I (16)mRNAs in the temporal cortex in AD. We found that thechanges of the ND15 mRNA level were proportional tothe changes of the ND4 mRNA level in three different

AD brain regions. Because of this the ratio betweenmtDNA-encoded ND4 mRNA and nDNA-encodedND15 was the same in all AD brain regions studied anddid not differ significantly from control values. ND isa one of the most complicated multi-subunit enzymecomplexes in the cell. Functions of seven mtDNA-encoded ND subunits are, in most part, unknown. How-ever, Hofhaus and Attardi (50) investigated the role ofthe ND4 subunit. These authors described a human cellline in which the ND enzyme complex lacks the ND4subunit due to a frameshift mutation in the gene. It wasobserved that the absence of ND4 polypeptide causedthe other mtDNA-encoded subunits to fail to assemble,while some of the nuclear-encoded subunits involved inredox reactions appear to assemble normally. As a re-sult, the normal ubiquinone oxidoreductase activity ofND was completely lost, but the Fe(CN)6 oxidoreduc-tase activity was normal. Thus, if the decrease of ND4mRNA in the AD brain observed in our study leads tothe same defect of ND enzyme complex, this defectmight be underevaluated in the studies, which measuredthe ND activity in AD using artificial electron acceptors.

It is unclear if the decreased activity of CO andother ETC enzymes is a primary defect in AD or this isthe consequence of the damage of mitochondria in de-generating neurons. Changes of the levels of mito-chondrial and nuclear-encoded mRNAs for subunits ofcomplex I and complex IV may reflect, in part, generalchanges of mitochondrial and nuclear transcription.Down-regulation of several mitochondrial transcripts,such as 16S and 12S RNA, ND6 mRNA, ATPase sub-unit 6 mRNA, CO I and CO III mRNAs, during oxida-tive stress was reported recently (51). The decrease ofmRNA production in the brain of AD patients due toincreased nuclear chromatin condensation also wasdocumented in several studies (52-55). Recently, wereported that several nuclear-encoded mRNAs, includ-ing "marker" beta-actin and cyclophilin mRNAs, maychange the same way as CO IV and ND15 mRNAs indifferent regions of the AD brain (56). The results ofour study suggest that the changes in the content ofmitochondrial and nuclear-encoded mRNAs forparts of ND and CO ETC complexes may contributeto the malfunction of oxidative metabolism in thebrain of AD patients.

ACKNOWLEDGMENTS

The authors thank Ms. Paula Thomason for assistance in manu-script preparation and Mr. Cecil Runyons for subject demographicdata. This work was supported in part by NIH grants AG-10836 (SE),

Expression of mRNAs for Cytochrome c Oxidase and NADH Dehydrogenase in Alzheimer's Disease

1 PO1-AG05119 (W.R.M.) by a grant from the Abercrombie Foun-dation (W.R.M.), and a grant from the Kleberg Foundation (W.R.M.).

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