Parkinsonism and Related Disorders 20S1 (2014) S68–S72

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Aldehyde dehydrogenase 2 in sporadic Parkinson’s disease b Tanja Maria Michel a , Ludwig Kasbauer ¨ , Wieland Gsell b , Julia Jecel c , Abigail Jane Sheldrick a , d e,f Miriam Cortese , Thomas Nickl-Jockschat a , Edna Grunblatt ¨ , Peter Riederer b,g, * a Department

of Psychiatry and Psychotherapy, Rostock University Medical School, Rostock, Germany Hospital Wuerzburg, Clinic and Policlinic for Psychiatry, Psychosomatics and Psychotherapy, University Wuerzburg, Germany c Department of Neurology, Danube Hospital, Center for Social Medicine East, Vienna, Austria d RWTH Laboratory for Plastic Surgery, RWTH Aachen University, Aachen, Germany e University Clinic of Child and Adolescent Psychiatry, University of Zurich, Zurich, Switzerland f Neuroscience Center Zurich, University of Zurich and ETH Zurich, Switzerland g Visiting Researcher at Department of Cognitive Brain Sciences, NCGG, Aichi, Japan b University

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Keywords: Aldehyde dehydrogenase 2 Parkinson’s disease Mitochondrial defect Oxidative stress Aldehyde toxicity

Aldehyde dehydrogenases (ALDH) play a key role in neuronal protection. They exert this function by metabolizing biogenic amine-related aldehydes, e.g. 3,4-dihydroxyphenylacetaldehyde (DOPAL), and by protecting neurons against aldehyde- and oxidative stress-related neurotoxicity. The role of these different isoenzymes has been discussed in other neurodegenerative disorders before. It is somewhat surprising that only few studies have investigated their role in the aetiology of Parkinson’s disease (PD), in both the degeneration of dopaminergic neurons and the formation of Lewy bodies. Earlier studies report severe alterations of the cytosolic isoform of ALDH expression (ALDH 1A1) in the substantia nigra of patients with PD. However, there are no data regarding the activity of ALDH 2 located at the inner mitochondrial membrane. Since mitochondrial dysfunctions are hypothesized to be of importance in the aetiology of PD we have examined the enzymatic activity of mitochondrial ALDH 2 in post-mortem putamen and frontal cortex of patients with PD and controls. We found that mitochondrial ALDH 2 activity in contrast to the frontal cortex was significantly increased in the putamen of patients with PD compared to controls. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Idiopathic Parkinson’s disease (PD) is a common neurodegenerative disorder characterised by a selective degeneration of neuromelanincontaining dopaminergic (DA) neurons in the pars compacta of the substantia nigra (SNpc) projecting to the putamen and the caudate nucleus of the brain. However, the process that leads to neurodegeneration in PD remains largely unknown. Evidence suggests that both environmental factors and genetic factors on the basis of e.g. mitochondrial dysfunctions, oxidative stress (OS) and aldehyde-related toxicity are major components accounting for the specific pathophysiology of PD [1]. OS propagates cell death by increased production of free radicals, which can be either reactive oxygen species (ROS) or reactive aldehyde species (RAS). A large number of studies suggest increased OS levels in patients with PD mediated by mitochondrial dysfunction, decreased concentrations of antioxidants and elevated iron levels [2]. These processes contribute to cell degeneration via lipid peroxidation. Oxidative * Corresponding author. Prof. Dr. Dr. hc. Peter Riederer, University Hospital Wuerzburg, Clinic and Policlinic for Psychiatry, Psychosomatics and Psychotherapy, Fuchsleinstraße ¨ 15, 97080 Wuerzburg, Germany. E-mail address: [email protected] (P. Riederer). 1353-8020/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.

deamination leads to the production of aldehydes which may render toxic if aldehyde dehydroxygenase (ALDH) activity is reduced. In fact, 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) have been found to be significantly increased in post-mortem SN of Parkinson’s disease. Both types of free radicals, namely ROS and RAS, can lead to cellular damage via the modification of proteins and inhibiting enzymes [3,4]. A significant increase of 4-HNE and MDA suggests that such aldehydes are not or insufficiently metabolised into acids by ALDH. ALDH isoenzymes differ in their affinity to various aldehydes and show an organ-specific distribution [5]. There are in particular two ALDH isoenzymes which play a role in detoxifying aldehydes in the brain, namely ALDH 1A1 and ALDH 2. ALDH 1A1 is located at the cytosolic site. Therefore it is in close vicinity to monoamine oxidase (MAO). ALDH 2 is located at the inner mitochondrial membrane. This demonstrates the importance of getting rid of toxic aldehydes both within the mitochondria as well as in the cytosol [6]. There is ample evidence that in PD MAO expression and function in the SN is not disturbed, while ALDH 1A1 is significantly reduced or even not present [1,7]. In fact, an increased ratio of 3,4-dihydroxyphenylacetaldehyde (DOPAL) to 3,4-dihydroxyphenylacetic acid (DOPAC) in the putamen of PD suggests decreased

T.M. Michel et al. / Parkinsonism and Related Disorders 20S1 (2014) S68–S72

detoxification of DOPAL by ALDH [8]. Elevated concentration of DOPAL correlates with increased protein modification. This has been demonstrated in experimental work in which DOPAL was added to proteins. The aldehyde reacts with Lys residues, yielding a Schiff base and finally protein cross-linking. Adverse cellular effects can be expected [9] as DOPAL is more than 1000-fold more toxic than dopamine (DA) in vivo [10]. There is a significant deficit of mitochondrial respiratory chain activity and especially complex I in the PD SN. Inhibition of complex I causes increases in DOPAL concentrations and death of DA neurons in vitro and in vivo as reviewed in [10]. This may be attributed at least in part to loss of ALDH 2 activity. Wey and colleagues [11] tested the hypothesis that chronically decreased function of multiple ALDHs caused by environmental toxins and/or reduced ALDH expression may play an important role in the pathophysiology of PD. They addressed this by generating mice null for ALDH 1A1 and ALDH 2. Those mice exhibited agedependent deficits in motor behaviour, significant loss of tyrosine hydroxylase positive neurons in the SN and a reduction of DA and metabolites in the striatum. DOPAL and 4-HNE were significantly increased [11]. Post-mortem studies show a marked reduction of ALDH 1 expression in surviving neurons on PD SNpc while there is no alteration in the ventral tegmental area [12]. Transcript profiling via quantitative RT-PCR in RNA originating from peripheral blood samples demonstrated significant gene changes in four genes including ALDH 1A1 [13]. Furthermore, gene expression profiling of sporadic PD SNpc reveals impairment of ubiquitin–proteasome subunits, SKP 1A and ALDH 1 [1]. Recent experimental work using knock down of SKP 1A rendered SN4741 cells sensitive to genetic reduction of ALDH 1 and external stressors [14]. While there is profound evidence that cytosolic ALDH 1A1 is lacking/reduced in PD SN, surprisingly enough there are no post-mortem data reflecting ALDH 2 activity. Therefore we have performed such analysis in order to improve understanding of mitochondrial disturbances in PD. 2. Material and methods 2.1. Brain tissue samples Human brain specimens were provided by the Brain Bank at the laboratory of Clinical Neurochemistry, Department of Psychiatry, University Hospital Wurzburg. ¨ The investigations were approved by the Committee for Medical Ethics at the University Hospital Wurzburg. ¨ All procedures were in accordance with the declaration of Helsinki as well as with the NIH Guideline for the Care and Use of Laboratory human tissue. The activity of ALDH 2 in the human brain was measured in postmortem brain tissue of 9 patients with PD (mean age: 76.4±8.9 years; 7 females, 2 males) and 12 matched control individuals (mean age: 80.7±7.4 years; 8 females, 4 males) without any psychiatric or neurological history. Control subjects and patients were carefully matched for post-mortem time, age and gender. There was no history of psychiatric illness or treatment with psychotropic drugs in any of the control subjects. Additional selection criteria for both patient and control groups was no lifetime history of alcohol or substance abuse or dependence. Patients and controls had a similar socio-economic background, and were of German-speaking Caucasian origin. Most individuals had died as a result of similar cardiovascular condition, pneumonia, sepsis or hepatic failure. The clinical notes reveal that all individuals had shown evidence of vascular conditions, such as the presence of one or more of the following: history of myocardial infarction, coronary artery disease, hypertensive cardiomyopathy or hypertension (see Table 1

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for sample characteristics). The patients fulfilled the diagnostic criteria of the international classifications of disease for PD. All patients have been in the final stages of their condition (Hoehn and Yahr stage: 5). Furthermore, a careful neuropathological postmortem examination showed significantly less pigmentation in the substantia nigra in patients with PD as well as Lewy-body pathology (Braak stage: VI). The brain regions investigated were the frontal cortex and putamen. 2.2. Tissue extraction Brain tissue was obtained according to a standardised procedure [15]. The left hemisphere was freshly frozen at −80°C. For further neuropathological investigations, the right hemisphere was kept in formalin for neuropathological examinations. Freshfrozen brain extracts were prepared by homogenisation in 10 volumes (w/v) of 0.25 M saccharine with a Polytron homogeniser (Cliffton, New Jersey) at 1000 rpm followed by sonification (Branson, Sonifier 250, Danbury) followed by centrifugation (600g for 10 min) at 4°C. The cellular debris was then re-suspended with 0.25 M sucrose, and the supernatant was centrifuged for another 10 min (15,000g, 4°C; Sorvall RC-5C). The cellular debris was then re-suspended again with 0.25 M saccharose and then sonified. The supernatants were centrifuged for another 60 min (100,000g, 4°C, Beckmann L3-50). The sediment with the microsomal fraction was again re-suspended with 0.25 M saccharose. 2.3. Protein quantification For protein quantification of the brain homogenate, the protein assay kit provided by BioRad on the basis of the method described by Bradford [16] was used. Colorimetric visualisation was carried out at 595 nm. 2.4. Mitochondrial ALDH 2 activity The mitochondrial ALDH 2 activity (nmol/min/mg protein) was determined using a colorimetric method modified according to the protocol described before [4,17]. Briefly, 50 mM sodium pyrophosphate buffer (pH 8.8), 0.5 mM NAD+, 1 mM EDTA, 10 mM 2-mercaptoethanol, 2 mM rotenone (1% of total volume), 0.5% Triton X-100 and freshly prepared 0.4 mg of the mitochondrial fraction were used as reactant, and the substrate indole-3-acetaldehyde (IAA) was added to start the reaction. 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 2 activity. The activity was calculated by both the standard curves and the extinction coefficient (Lambert–Beer law). 2.5. Statistics We compared the ALDH 2 activities between patients with PD and age-matched controls using Student’s t-test on the statistical software GraphPad Prism version 4. The influence of independent variables such as gender, age and post-mortem delay was tested using covariance analysis (ANCOVA). In addition, we examined the relation of age and ALDH 2 activity by performing Pearson Product Moment Correlation. Significance was set at a p-value of 0.05

Putamen

5.9±0.45

7.52±0.46