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Increased Activity of Mitochondrial Aldehyde Dehydrogenase(ALDH) in the Putamen of Individuals with Alzheimer's

Disease: A Human Postmortem Study

Michel, T M; Gsell, W; Käsbauer, L; Tatschner, T; Sheldrick, A J; Neuner, I;Schneider, F; Grünblatt, E; Riederer, P

Michel, T M; Gsell, W; Käsbauer, L; Tatschner, T; Sheldrick, A J; Neuner, I; Schneider, F; Grünblatt, E; Riederer,P (2010). Increased Activity of Mitochondrial Aldehyde Dehydrogenase (ALDH) in the Putamen of Individualswith Alzheimer's Disease: A Human Postmortem Study. Journal of Alzheimer's Disease:Epub ahead of print.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:Journal of Alzheimer's Disease 2010, :Epub ahead of print.

Michel, T M; Gsell, W; Käsbauer, L; Tatschner, T; Sheldrick, A J; Neuner, I; Schneider, F; Grünblatt, E; Riederer,P (2010). Increased Activity of Mitochondrial Aldehyde Dehydrogenase (ALDH) in the Putamen of Individualswith Alzheimer's Disease: A Human Postmortem Study. Journal of Alzheimer's Disease:Epub ahead of print.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:Journal of Alzheimer's Disease 2010, :Epub ahead of print.

Increased Activity of Mitochondrial Aldehyde Dehydrogenase(ALDH) in the Putamen of Individuals with Alzheimer's

Disease: A Human Postmortem Study

Abstract

For decades, it has been acknowledged that oxidative stress due to free radical species contributes to thepathophysiology of aging and neurodegenerative diseases. Aldehyde dehydrogenases (ALDH) not onlytransform aldehydes to acids but also act as antioxidant enzymes. However, little is known about theimplications of the enzymatic family of ALDH in the context of neurodegenerative processes such asAlzheimer's disease (AD). We therefore examined the enzymatic activity of the mitochondrialALDH-isoform in different regions of the postmortem brain tissue isolated from patients with AD andcontrols. We found that the mitochondrial ALDH activity was significantly increased only in theputamen of patients suffering from AD compared to controls. This is of particular interest sincemediators of oxidative stress, such as iron, are increased in the putamen of patients with AD. This studyadds to the body of evidence that suggests that oxidative stress as well as aldehyde toxicity play a role inAD.

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Journal of Alzheimer’s Disease ISSN: 1387-2877 IOS Press DOI: 10.3233/JAD-2009-1326

Increased Activity of Mitochondrial Aldehyde Dehydrogenase (ALDH) in the Putamen of

Individuals with Alzheimer’s Disease: A Human Postmortem Study

Tanja Maria Michela,b,c, Wieland Gselld, Ludwig Käsbauerb, Thomas Tatschnere, Abigail Jane

Sheldricka, Irene Neunera, Frank Schneidera,c, Edna Grünblattb and Peter Riederera

aDepartment of Psychiatry and Psychotherapy, RWTH Aachen University, Aachen, Germany

bClinical Neurochemistry, University Clinic & Policlinic Psychiatry, Psychosomatic and

Psychotherapy, University of Würzburg, Würzburg, Germany

cJARA – Translational Brain Medicine, Aachen, Germany

dState Hospital for Psychiatry, Psychotherapy and Neurology, Lohr am Main, Germany

eDepartment of Neuropathology, Institute of Pathology, University of Würzburg, Germany

Running title: Mitochondrial ALDH in Alzheimer’s disease

Accepted 1 November 2009

Correspondence to: Tanja Maria Michel, MD, Department of Psychiatry and Psychotherapy,

RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany; Tel:+492418036258,

Fax:+492418082524, Email: tmichel@ukaachen.

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ABSTRACT

For decades, it has been acknowledged that oxidative stress due to free radical species

contributes to the pathophysiology of aging and neurodegenerative diseases. Aldehyde

dehydrogenases (ALDH) not only transform aldehydes to acids but also act as antioxidant

enzymes. However, little is known about the implications of the enzymatic family of ALDH in

the context of neurodegenerative processes such as Alzheimer’s disease (AD). We therefore

examined the enzymatic activity of the mitochondrial ALDH-isoform in different regions of the

postmortem brain tissue isolated from patients with AD and controls. We found that the

mitochondrial ALDH activity was significantly increased only in the putamen of patients

suffering from AD compared to controls. This is of particular interest since mediators of

oxidative stress, such as iron, are increased in the putamen of patients with AD. This study adds

to the body of evidence that suggests that oxidative stress as well as aldehyde toxicity play a role

in AD.

Keywords: aging, aldehydehydrogenase, Alzheimer’s disease, dementia, free radicals,

mitochondria, oxidative stress

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INTRODUCTION

In Alzheimer’s disease (AD), increased toxic damage to the brain due to increased reactive

aldehydes (RA) has been associated with neuropathological hallmarks of the disease such as

neurofibrillary tangles and neuritic plaques [1]. RA are highly reactive transient molecules,

which are induced by endogenous and exogenous processes (Figure 1a-c). If not properly

degraded, RA target cell membranes and induce lipid peroxidation, modify enzyme function,

inducing mitochondrial dysfunction, and induce oxidative stress, which is also mediated by

increased cerebral iron, leading to apoptosis [2]. Both oxidative stress and aldehyde toxicity have

been acknowledged to play a key role in the pathophysiology of neuropsychiatric disorders and

AD via lipid peroxidation and DNA strand brakes [3-12]. Oxidative stress refers to an imbalance

within cells, where more reactive oxygen species (ROS) are produced than are detoxified,

leading to cell damage and death [13,14].

One of the most important pathways for aldehyde detoxification is their oxidation by

aldehyde dehydrogenase (ALDH, [EC 1.2.1.3.]). ALDH is a well known enzyme with pyridine-

nucleotide-dependent oxidoreductase activity which is important for the metabolism of aldehyde

substrates in numerous species [15].

The ALDH superfamily includes NAD(P)+-dependent enzymes catalyzing the oxidation of a

wide spectrum of aliphatic and aromatic aldehyde substrates generated from various endogenous

and exogenous precursors to their corresponding carboxylic acids. Endogenous aldehydes are

formed during the metabolism of vitamins, steroids, lipids, amino acids, neurotroansmitters,

biogenic amines and in the process of lipid peroxidation (Figures 1b,c). Exogenous aldehydes are

formed during the metabolism of numerous agents such as alcohol, drugs and environmental

agents (Figure 1a).

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In AD, one special focus has been on one of the isoenzymes, the mitochondrial ALDH

(ALDH2). This isoenzyme of ALDH is located in the mitochondrial matrix and is considered to

be principally responsible for the oxidation of acetaldehyde which is produced during different

endogenous and exogenous processes mentioned above. It is involved in the metabolism of

biogenic aldehydes produced by monoamines and oxidase-catalyzed deamination of

catecholamines and indoleamines [15-17]. Furthermore, dicarbonyl compounds such as

methylglyoxal or glyoxal are also substrates for ALDH. These compounds have been implicated

in the pathogenesis of AD [18,19].

A functional polymorphism of the mitochondrial aldehyde dehydrogenase gene (ALDH2 1/2

polymorphism) can influence the accumulation of acetaldehyde which may play a role in

Alzheimer’s disease (AD) and has a high prevalence among Mongoloids [20-22]. The results of

the association studies were varying among different Asian subpopulations. An association with

late-onset AD (LOAD), interacting synergistically with the presence of the apolipoprotein E

allele 4 (APOE ε4) has been detected in Japanese and Chinese subjects, whilst it was not

reported in a population of Koreans [20-24]. A longitudinal study found that the incidence of AD

in healthy participants was not correlated with ALDH polymorphism within 2.4 years [24]. In the

mouse model, it has been shown that a deficiency in mitochondrial ALDH2 leads to age

dependent memory impairment and increased lipid peroxidation [25]. Furthermore, these animals

show the same neuropathology, namely hyperphosphorylation as well as plaques and tangles, as

seen in humans with AD [25].

It has been suggested that mitochondrial ALDH influences the pathophysiology of AD in

several ways. First of all, it influences aldehyde toxicity directly, secondly it significantly

contributes the overall antioxidative potential on a cellular level, and thirdly it interferes with

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various neurotransmitters that are decreased in the context of AD. All these different

mechanisms are well known to play a pivotal role in AD. However, to this date, mitochondrial

ALDH is not one of the well-studied antioxidant players that scientists expected to find fighting

free-radical damage. Little is known about the activity and function of this enzyme in the brain

of patients with AD [26]. For this reason, it is worth examining the activity of mitochondrial

ALDH more closely in this context. We have therefore studied the enzyme activity of

mitochondrial ALDH in the cerebral cortex as well as the putamen of patients with AD since

both regions are known to play a role in AD and have an increased iron concentration, which

mediates oxidative stress in both aging and AD [27-29].

MATERIALS AND METHODS

Brain tissue samples

The activity of the mitochondrial aldehydedehydrogenase, ALDH, in the human brain was

measured in postmortem brain tissue of 14 elderly patients with mostly final stages of AD (9

females, 5 males, mean age: 81.6±4.4 years) and 12 age-matched controls (8 females, 4 males,

mean age: 80.8±4.4 years) without any medical history of psychiatric or neurological diseases.

Two of the age-matched controls showed neurofibrillary tangles and senile plaques. The patients

fulfilled the diagnostic criteria of the International classifications of disease for AD [30].

Additionally, the diagnosis was verified via postmortem examination for senile plaques and

neurofibrillary tangles [31]. None of the brains examined had signs of other neurodegenerative

diseases such as Parkinson’s disease (PD). The brain regions investigated were the frontal cortex

and the putamen. Brain samples from Caucasian German speaking patients were matched with

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control samples according to age, gender, and postmortem delay (See Tables 1, 2, and 3 for

details).

The postmortem brain tissue was collected via autopsies performed at the Department of

Psychiatry, Hospital, Mauer, Austria and at the Institute of Forensic Medicine, University of

Würzburg, Germany. All procedures were in accordance with the NIH Guide for the Care and

Use of Laboratory human tissue and were approved by the Ethics committee of the University of

Würzburg, Germany and were also in accordance with the declaration of Helsinki.

Tissue extraction

The brain tissue was obtained according to a standardized procedure [32]. The left

hemisphere was freshly frozen at -80°C. For further neuropathological investigations, the right

hemisphere was kept in formalin. Freshly frozen brain extracts were prepared by homogenization

in 10 volumes (w/v) of 0.25 M saccharine with a Polytron homogenizer at 1000 rpm followed by

sonification (Branson). After this procedure, centrifugation (600g for 10 min) was carried out for

10 min at 4°C. The cellular debris was then re-suspended with 0.25M sucrose and the

supernatant was centrifuged for another 10 min (15000g at 4°C; Sorvall RC-5C). The cellular

debris was then re-suspended again with 0.25M saccharine and then sonified. The supernatants

were centrifuged for another 60 min (100,000g, 4°C Beckmann L3-50). The sediment with the

mitochondrial fraction was re-suspended with 0.25M.

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Protein quantification

For protein quantification of the brain homogenate, the protein assay kit provided by BioRad

on the basis of the method previously described [33] was used. Colorimetric visualization was

carried out at 595 nm.

Mitochondrial ALDH Activity

The mitochondrial ALDH activity (nmol/min/mg protein) was determined using a

colorimetric method modified according to the protocol described before [34]. Briefly, 50 mM

sodiumpyrophosphate-buffer (pH 8.8), 0.5 mM NAD+, 1 mM EDTA, 10 mM 2-

Mercaptoethanol, 2 µM Rotenon (1% of total volume), 0.5 % Triton X-100 and freshly prepared

homogenate were used as reactant and the substrate indol-3-acetaldehyde (IAA) was added to

start the reaction. 1 mg of the brain homogenate and 0.4 mg of the mitochondrial fraction were

added to the reagent-substrate mixture. We later omitted the mitochondrial fraction to calculate

the result for the cytosolic fraction (data not shown). The change in optical density at 340 nm

(37°C) was measured over time (20 min) by a spectrophotometer (552S UV/VIS; Perkin-Elmer,

USA) to obtain the ALDH activity (Lambert-Beer law). The activity was calculated by the PC

connected to the photometer using both the standard curve and the extinction coefficient

(Lambert-Beer law).

Statistics

We compared the different ALDH activities between the index-group (patients with AD) and

age matched control-group using student’s t-test on the statistical software graph-pad-prism

version 4.

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The influence of independent variables such as gender, age, and postmortem delay was tested

using covariance analysis (ANCOVA). In addition, we examined the relation of age and ALDH

activity by performing Pearson Product Moment Correlation. Significance was set at p-value <

0.05.

RESULTS

The mitochondrial ALDH activity (mean±SEM in mmol/min/mg protein) was significantly

(p<0.029, df=22) elevated in the putamen of the AD group (7.60±0.54, 22) compared to the

control group (5.9±0.45) (See Figure 2). In the frontal cortex, we were not able to detect any

significant differences between the groups (AD: 8.29±0.49; controls: 8.07±0.43, p >0.74, df=24).

The effect size for the putamen was 0.2 and for the frontal cortex 0.13.

Although patients and controls were carefully pair-matched, we examined the influences of

confounding parameters such as age, gender, medication, and postmortem delay on ALDH

activities. There was no significant correlation with postmortem time (0.41<rho<0.36;

0.19<p<0.96) nor age (-0.49<rho<0.52; 0.11<p<0.94) in any of the investigated brain regions.

DISCUSSION

In our study, we found that mitochondrial ALDH-activity was significantly higher in the

putamen of patients with AD. Our results are in line with previous studies suggesting a role of

ALDH in the pathogenesis of AD [20-23,26].

Our results of an alteration of the mitochondrial ALDH activity in the brain of patients with

AD are supported by earlier findings of Picklo and colleagues [26] who also found the ALDH

activity to be significantly elevated in the brain of patients with AD. This group reported a

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significant increase of ALDH in the temporal cortex of patients with AD in a significantly

smaller sample of only four patients with AD [26]. To the best of our knowledge, we are the first

to describe an increase of mitochondrial ALDH in the putamen of a patient with AD. This study

suggested that increased mitochondrial ALDH expression might be due to increased highly

reactive intermediates or pro-oxidants in AD, since mitochondria are one of the main sources of

free radicals in organs [26]. Earlier studies on ALDH in AD have suggested that increased

ALDH activity, as we found in the putamen, might play a main role in the formation of senile

plaques [26].

A number of reasons could be responsible for the increased mitochondrial ALDH activity

that we report in the putamen of patients with AD. First, it might be a result of increased

production of aldehydes due to an increase of monoamine oxidase B (MAO-B), which has been

reported in the context of AD [35]. Furthermore, the increased ALDH activity, together with an

elevated MAO-B activity, could be responsible for a higher dopamine turnover and an increased

oxidative stress which has been found in the putamen of patients with AD [35-37]. The latter

induces MAO-B activity, via a positive feed-back loop and in turn increases oxidative stress and

neurodegeneration [37,38].

Second, in the context of AD, serotonergic, dopaminergic, and noradrenergic neurons

degenerat, due to multiple factors, e.g., disruption in neurotrophic factors, and increased iron

mediated oxidative stress [37,39,40]. These neurodegenerative processes warrant an increase of

the metabolizing enzymes, e.g., aldehyde dehydrogenases AD [41].

Thirdly, it has been reported that the putamen in particular contains large quantities of iron

which increase with age [29]. Furthermore, in AD, iron deposits in the putamen have been

reported to be significantly elevated in subjects with cognitive deficits [27,28]. Since iron plays a

10

pivotal role in oxidative stress, increased iron content in the putamen of patients with AD could

contribute to elevated oxidative stress, which in turn could increase both ROS and RAs. Both

could then lead to neurodegeneration which is described in the context of AD [30,39,42]. The

increase of RA might warrant metabolism catalyzed by increased mitochondrial ALDH [41].

However, we did not measure the iron content of the putamen in our study.

Finally, several studies suggest that alcohol intake affects the development of AD [43],

because ethanol and its metabolite, acetaldehyde, are directly neurotoxic. Therefore, a

dysregulation of ALDH leading to a disruption of the ethanol mechanism resulting in increased

aldehyde toxicity might contribute the development of AD in the context of increased alcohol

intake (ADH) [43]. However, since none of our AD subjects had a significant history of alcohol

addiction, external ethanol intake might play only a minor role in the increase of mitochondrial

ALDH that we found in the putamen of the index group.

Similar to what we observed with ALDH, increased expression of enzymes to detoxify ROS

have been reported earlier in AD. One example is manganese superoxide dismutase (Mn-SOD)

which is localized in the mitochondria and catalyzes the detoxification of superoxide [36].

We did not find a significant correlation with either the postmortem time or age and ALDH

activity. This is suggestive of a high postmortem stability of the enzyme as has been reported

before [44].

Our study is the first to report a significantly increased mitochondrial ALDH activity in the

putamen of patients with AD. However, the role of neuroprotection and degeneration in AD

remains complex since neuroprotective agents such as ALDH, neurotrophic factors, and their

interaction with oxidative stress in AD play an important role and have to be examined in further

studies [39,40,45].

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The significance of our current examinations is highlighted by a recent study showing that

treatment with methylene blue (methylthioninium chloride) may postpone or reverse

neurodegeneration in AD [46]. Methylene blue inhibits tau aggregation and works as a hydrogen

ion scavenger, an inhibitor of nitric oxide synthase as well as an inhibitor of the enzyme MAO

[47]. Both pathomechanisms are linked to oxidative stress as well as aldehyde toxicity and the

function of ALDH. Therefore, the therapeutic effect of methylene blue may be linked to its

ability to reduce oxidative stress and or aldehyde toxicity.

It is pivotal to carry out further studies linking possible mutations of the ALDH genes and

treatment outcome in AD. This has been carried out previously for other diseases that are linked

to ALDH and lead to neuronal damage or loss such as alcohol dependence [48]. Thus, the

metabolism of aldehyde, including the key enzyme mitochondrial ALDH, could be a new target

for preventive and therapeutic strategies in AD. However, so far, there are only few studies on

the subject and further investigations are needed for a better understanding of the complicated

interactions of this family of enzymes, namely ALDH, to explore future therapeutic implications.

DISCLOSURE STATEMENT

Authors’ disclosures available online (http://www.j-alz.com/disclosures/view.php?id=195).

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Table 1. Sample characteristics and activity of ALDH in the frontal cortex and the putamen. Number Age Gender

F/M Postmortem Cortex

frontalis p Putamen p

Control 12 80.8±7.5 8/4 8.2±5.1 8.07±0.43 5.9±0.45 AD 14 81.6 ± 4.4 9/5 9.4±9.5 8.29±0.49

>0.7 7.60±0.54

<0.03

20

Table 2. Demographic and neuropathological data of patients with AD Diagnosis Age Sex PM Cause of death Neuropathology/

Stage of disease AD 81 F 18 Pneumonia

Cardiac arrest Cerebral atrophy, Final stage of AD

AD 82 M 12 Pneumonia Cardiac arrest

Final stage AD

AD 83 M 6 Pulmonary embolism Cerebral Atrophy, internal hydrocephalus Final stage AD

AD 80 F 15 Sepsis Cerebral Atrophy Final stage AD

AD 80 F 4.5 Pulmonary embolism Cerebral Atrophy Final stage AD

AD 72 F 3 Sepsis

Cerebral Atrophy, micro-vascular cerebral infarction,

AD AD 86 F 38 Pulmonary embolism Diffuse cerebral Atrophy

AD (NS) AD 86 M 9 Pneumonia Diffuse cerebral Atrophy

AD (NS) AD 77 M 3.5 Cardiac arrest Atrophy

Cerebral arteriosclerosis AD

AD 88 F 3 Pulmonary embolism Diffuse cerebral Atrophy Final stage AD

AD 84 F 5 Cardiac arrest Diffuse cerebral Atrophy Final stage AD

AD 76 M 6 Pneumonia, Cardiac

arrest Cerebral Atrophy Final stage AD

AD 84 F 5 Pneumonia, Cardiac arrest

Cerebral Atrophy Final stage AD

AD 83 F 3 Ileus Cerebral Atrophy Final stage AD

21

Table 3. Demographic and neuropathological data of controls. Group Age Sex PM Cause of death Neuropathology

C 80 F 3.00 Pulmonary embolism - C 87 F 15.00 Cardiopmopathy Regular aged brain with a few

fibrillary tangles C 78 F 12.00 Pulmonary embolism

Cardiac arrest -

C 88 M 10.50 Pneumonia, Pulmonary embolism

-

C 72 F 3.00 Pulmonary embolism - C 85 F 13.00 Cardiac arrest - C 80 M 5.10 Acute cardial

dilatation, stroke Regular aged brain with a few

fibrillary tangles C 90 M 17.00 Cardiac arrest,

Pneumonia -

C 86 F 5.50 Pulmonary embolism - C 63 F 6.50 Bronchial carcinoma,

metastasis -

C 80 F 4.00 NK - C 80 M 3.50 Cardiac arrest

Sepsis -

22

FIGURE LEGEND

Figure 1. (a,b,c) The metabolism of ALDH, an exogenous ethanol-metabolism; (b)

neurotransmitter metabolism; (c) ALDH is detoxifying toxic aldehydes in the lipid peroxidation

process.

23

Figure 1