Genomic aspects of sporadic Parkinson\'s disease

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Parkinsonism and Related Disorders xx (2008) 1e4 www.elsevier.com/locate/parkreldis

Genomic aspects of sporadic Parkinson’s disease* P. Riederer a,*, M.B.H. Youdim b, S. Mandel b, M. Gerlach c, E. Gru¨nblatt a a

Institute of Clinical Neurochemistry and National Parkinson Foundation Centre of Excellence Laboratories, Clinic and Policlinic for Psychiatry and Psychotherapy, University of Wu¨rzburg, Fu¨chsleinstr. 15, D-97080 Wu¨rzburg, Germany b Eve Topf and NPF Centers of Excellence for Neurodegenerative Diseases Research, Technion-Rappaport Family Faculty of Medicine, P.O. Box 9649, IL-31096 Haifa, Israel c Laboratory of Clinical Neurochemistry, Clinic for Child and Adolescent Psychiatry and Psychotherapy, University of Wu¨rzburg, Wu¨rzburg, Germany

Abstract Parkinson’s disease (PD) is thought to be associated with oxidative stress mechanisms, as well as with glutamate receptor abnormalities, ubiquitineproteasome dysfunction, inflammatory and cytokine activation, dysfunction in neurotrophic factors, damage to mitochondria, cytoskeletal abnormalities, synaptic dysfunction and activation of apoptotic pathways. To investigate these hypotheses, many researchers have applied molecular biology techniques to the study of neuronal cell death in these conditions. In this article, we discuss recent findings of gene expression in PD that may elucidate the usage of specific new biomarkers for sporadic PD and point to novel drug developments. Ó 2008 Published by Elsevier Ltd. Keywords: Affymetrix; Gene expression; 6-Hydroxydopamine; Methamphetamine; Microarray; MPTP; Parkinson’s disease; Quantitative RT-PCR

1. Introduction Central genetic dogma states that genomic DNA is first transcribed into mRNA, after which mRNA is translated into proteins. Proteins are critical for a wide range of intra- and extracellular activities, including enzymatic, regulatory and structural functions. Estimates suggest that 50% of the human transcriptome, the collection of all mRNAs in a cell, is expressed in the brain. Changes in mRNA expression can result in phenotypical and morphological modifications. Alterations in patterns of expression of multiple genes can offer new data concerning the existence of regulatory mechanisms and biochemical pathways. Therefore, the study of mRNA expression patterns in neurodegenerative disease may reveal the involvement of mechanisms of oxidative stress. Various methods can be used to measure gene expression at the *

This article is based on a presentation given at the LIMPE Seminars 2007 ‘‘Experimental Models in Parkinson’s Disease’’ held in September 2007 at the ‘‘Porto Conte Ricerche’’ Congress Center in Alghero, Sardinia, Italy. * Corresponding author. Tel.: þ49 931 201 77210; fax: þ49 931 201 77220. E-mail address: [email protected] (P. Riederer).

mRNA level, such as northern blot, polymerase chain reaction (PCR) after reverse transcription (RT), nuclease protection, cDNA sequencing, clone hybridization, differential display (DD), serial analysis of gene expression (SAGE) and microarray technology. These methods allow the characterization of several profiles of global gene expression, as well as screening for significant differences in mRNA abundance [1]. In the past five years, transcriptomics and proteomics have become the most preferred methods for large-scale gene expression assessment after DD and SAGE techniques. Although these methods are more elaborate compared with microarrays, they provide the possibility of revealing novel unknown genes. The study of global gene expression allows us to gain an understanding of the mechanisms of neuronal cell death in many diseases such as Parkinson’s disease (PD), Alzheimer’s disease, amyotrophic lateral sclerosis and prion disease. In these studies, mechanisms common to many neurodegenerative disorders have been revealed, such as neuro-inflammatory cascades, oxidant as well as antioxidant proteins, apoptotic as well as anti-apoptotic cascades, neuronal differentiation, synaptic trafficking mechanisms, transcription signalling and ubiquitinproteasomal cascades. These studies were carried out in

1353-8020/$ - see front matter Ó 2008 Published by Elsevier Ltd. doi:10.1016/j.parkreldis.2008.04.009 Please cite this article in press as: Riederer P et al., Genomic aspects of sporadic Parkinson’s disease, Parkinsonism and Related Disorders (2008), doi:10.1016/ j.parkreldis.2008.04.009

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post-mortem tissues of patients with neurodegenerative disorders, as well as in brain areas of animal models and in neuronal cell culture [2]. In this review, we discuss recent findings on gene expression of the substantia nigra pars compacta (SNpc) from post-mortem sporadic PD patients affected by degeneration of dopamine neurons. 2. Human post-mortem Parkinson’s disease The study of post-mortem tissues is made difficult [3,4] by the scarce availability of brain tissues. Thus, only very recently have several research groups managed to study gene expression in human PD patients. However, comparison of results produced by different groups is made difficult by variations in the preparation of the substantia nigra (SN) tissue. The ideal solution to this would be an international agreement to use exactly similar regions and specifically the SNpc where neuromelanin-containing dopamine neurons are located. The first study on gene expression profiles was carried out on the SN and adjacent midbrain tissues of two normal patients using SAGE and revealed 402 SN genes within five large genomic linkage regions [5]. Genes such as transcription elongation factor A (SII)-like 1, apolipoprotein J, ferritin heavy polypeptide 1 and beta-2-microglobulin were amongst the 20 most highly expressed SAGE tags in the pooled SN libraries. However, no comparison with PD tissue was conducted in this study. Our research group was able to show, for the first time, specific gene expression patterns in post-mortem SNpc of sporadic PD patients using Affymetrix GeneChip arrays [6]. This study identified decreased expression of 68 genes and enhanced expression of 69 other genes in the SNpc of PD patients compared with controls. Classification into functional groups revealed that genes related to signal transduction, protein degradation (e.g., ubiquitineproteasome subunits), dopaminergic transmission/metabolism, iron transport, protein modification/phosphorylation and energy pathways/ glycolysis functional classes are downregulated. Decreased expression of five subunits of the ubiquitineproteasome system, SKP1A (a member of the SCF (E3) ubiquitin ligase complex), and chaperone HSC-70, which can lead to severe functional impairment of an entire repertoire of proteins, was found in PD patients. Genes involved in adhesion/cytoskeleton, extracellular matrix components, cell cycle, protein modification/phosphorylation, protein metabolism and transcription, and inflammation/hypoxia (e.g., key iron and oxygen sensor EGLN1) were upregulated in PD SNpc. These results have shed light on the molecular mechanisms implicated in PD and may explain how misfolded proteins and ubiquitinated substrates accumulate and aggregate in Lewy bodies, such as a-synuclein, synphilin-1 and tyrosine hydroxylase, or in neurofibrillary tangles, such as tau-proteins and amyloid plaques with amyloid-beta in both genetic and sporadic diseases. The analysis of gene expression in homogenates of human post-mortem brains does not allow cell type-specific interpretations. For such studies, more sophisticated methodologies are required. Single-cell analysis, using laser capture

microdissection technique, can attain such a specificity as shown by Lu et al. [7]. These authors were able to generate specific RNA fingerprints with high resolution from phenotype-specific single neurons. Using DD, the same research group compared mesencephalic dopaminergic neurons containing Lewy bodies with neurons void of Lewy bodies in PD patients [8]. Their analysis demonstrated that 64 expressed sequence tags were altered in the dopaminergic neurons containing Lewy bodies. One of the genes found to be upregulated in the Lewy body-containing neurons is the ubiquitin specific peptidase 8 (USP8). As in the previous study, this observation points to the involvement of protein misfolding and degradation in PD. The stress 70 protein chaperone, microsomeassociated, 60 kDa (STCH) showed downregulation in the Lewy body-containing neurons. This protein is known to be a ‘‘core ATPase’’ encoding Hsp70-like gene, which was also shown to be altered in the first GeneChip analysis. In a further human post-mortem study of controls [9], a comparison was made between the nigrosome within the SNpc prone to degenerate in PD and the central grey substance, which is resistant to the disease. Immunolaser-microdissection-captured neurons were identified by anti-tyrosine hydroxylase quick immunostaining and melanization. RNA fingerprinting was performed according to Lu et al. [7]. Dopamine neurons in the central grey substance expressed genes of interest in cell survival (C7 fragment, ANP32B, NPM, PD41, KLHL1, NRG1, USP8), whereas SNpc dopaminergic neurons expressed the gene APLP2, known to be of importance in cell-death mechanisms. These data demonstrate the existence of regional differences in gene composition and gene expression, which are important to improve understanding of cell-death sensible regions and give limits for neuroprotective strategies based on both genetic targets and pharmacological manipulation of gene expression profiling. Similar studies have been performed in C 57 B1/6 mice comparing tyrosine hydroxylase-positive neurons of the A9 area with those of the A10 area. In the A9 area, GIRK2, ANT-2 and IGF-1 were increased compared with those in the A10 area, whereas in the A10 area there were three elevated peptides, GRP, CGRP and PACAP, compared with those in the A9 area [10]. Such data clearly demonstrate two important points: (1) there are region-dependent gene expression profiles; and (2) there is no identity of A9 dopaminergic neuron gene expression profiles between C 57 B1/6 mice [10] and post-mortem human SNpc nigrosome profiles [9]. Zhang and colleagues [11] studied gene expression alterations in three different brain regions of PD patients (the SN, putamen and area 9) using Affymetrix GeneChip arrays. A striking similarity to our results was found in all three brain regions. Upregulation of heat shock 27-kDa protein 1 (HSPB1) mRNA and downregulation of the NADH dehydrogenase (ubiquinone) FeeS protein 1 (NDUFS1) mRNA, SKP1 family tetramerization domain, a-synuclein and synaptosomal-associated protein, 25 kDa (SNAP25) were observed. Hauser et al. [12] compared substantia nigra gene expression in four different groups (PD, controls, progressive supranuclear palsy and fronto-temporal dementia with parkinsonism) using Affymetrix GeneChip arrays. They found 142 genes

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Fig. 1. Schematic diagram of major gene and neurochemical alterations in human (SNpc) of PD. Gene expression analysis of SNpc of PD has confirmed and extended the previously established complexities by which dopaminergic neurons degenerate. These findings do not allow a conclusion to be reached regarding the primary biochemical event(s) that induces the ‘‘domino’’ death cascade, resulting from excessive generation of nitric oxide/peroxynitrite (NO/ONOO), O2 or hydroxyl radicals, can lead to dysregulation of iron metabolism, induction of a-synuclein aggregation and mitochondrial dysfunction. Free (labile) iron itself can cause OS, aggregation of a-synuclein and degradation of iron regulatory protein 2 (IRP2) via activation of egl nine homologue 1 (Caenorhabditis elegans) (EGLN1), which is a key iron and OS sensor. This in turn results in proteasomal degradation of hypoxia-inducible factor (HIF) and IRP2, with subsequent decreases in cell survival/proliferation, glucose and iron metabolism genes. At the same time, an increase in gene expression of HIF-1 responsive RTP801 was found in PD SN. Increases in the expression of cell adhesion molecules and components of the extracellular matrix in response to OS/free radicals can result in cell assembly disruption. Aldehyde derivatives of dopamine metabolism are highly neurotoxic and aldehyde dehydrogenase (ALDH) is the key enzyme for their metabolism to inert acidic metabolites (homovanillic acid and dihydroxyphenylacetic acid). The reduction in gene expression of ALDH1A1, ARPP-21 and VMAT2, which are located within dopamine-containing neurons of SNpc, may contribute to a failure in DA transmission and metabolism. Significant evidence has been provided for involvement of protein misfolding in dopamine neuronal death. SKP1A is part of the SCF (SKP, Cullin, F-box protein) ubiquitin ligase component (E3) that regulates normal degradation of a wide array of proteins, which may include a-synuclein, parkin, IRP2, HIF, etc. Its decline can cause evasion of proteins subjected to SCF/26S proteasome complex degradation. This protein processing is exacerbated if some of the 26S proteasome subunits (PSM) are downregulated, as observed in several studies, since they are an integral part of the regulatory and catalytic activity of the proteasome. In addition, the ubiquitin activating enzyme-1 (UBE1) seems to alter its expression, which may influence the SCF process. The decreased expression of the chaperone HSC-70 may affect the correct folding of several proteins that are specifically ubiquitinated by the co-chaperone carboxyl terminus of HSC-70 interacting protein (CHIP), as well as parkin-CHIP-mediated ubiquitination, and may increase aggregation of a-synuclein and iron-induced OS. Another protein involved in this processes was found to have a decreased expression in PD: the UCHL1 or so-called PARK5 (ubiquitin carboxyl-terminal esterase L1). In addition, the mitochondrial dysfunction that is also known in the MPTP model for PD was supported by the decreased expression of several NADH dehydrogenase proteins (NDU). Red boxes are for upregulated genes, and blue boxes are for downregulated genes. Sharp arrows indicate positive inputs, and blunt arrows are for inhibitory inputs. (Modified from Gru¨nblatt et al. [6].).

significantly altered between PD cases and controls, and 96 genes significantly altered between progressive supranuclear palsy and fronto-temporal dementia with parkinsonism and controls. There were 12 genes common to all disorders. Again, as in all other three studies, four pathways were shown to be altered in PD SN substantia nigra: chaperones, ubiquitination, vesicle trafficking and nuclear-encoded mitochondrial genes. These new findings may help to develop new hypotheses concerning the aetiology of the disease and may provide a better diagnostic tool for clinicians. We have assembled these findings into a cascade of events which may show the relationship between oxidative stress and these gene alterations and cell death (Fig. 1). The reasons for the existence of discrepancies between (a) various gene expression studies performed with human postmortem brain tissue and (b) those data obtained from studies

with rodents and human post-mortem brain tissue have been reviewed recently [13]. Tables 1 and 2 show the factors that may be responsible for such discrepancies. The technology of gene expression profiling is a discovery-based procedure and therefore it is highly suitable for the study of idiopathic and multifactorial diseases. However, when several strategies Table 1 Confounding clinical factors influencing gene expression profiling Pathological stress Pre-mortem clinical evaluation Agonal state Chronic medication Time course of disease Variation in patient polymorphism profiling

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Table 2 Confounding technical factors influencing gene expression analyses Post-mortem factors RNA integrity/RNA quality testing Tissue preservation Single-cell vs cell mixture analysis Heterogeneity of cells of interest Small numbers of cells representative of population of cells of interest? Microarray platforms Quality of microarrays Microarray data analysis Gene target validation Whole tissue vs single-cell gene arrays in the same biological samples is missing Multiregional approach to be used Genotype-specific studies needed

of gene expression profiling are being compared in the same tissue sample, it becomes evident that this technology is still under development (R. Reynolds, BrainNet Europe II [BNEII] validation experiments, unpublished data). Furthermore, BNEII-based studies will be able to answer the question of how clinical factors influence gene expression analysis. 3. Conclusion In conclusion, studies of gene expression alterations reveal a wide range of pathways involved in neurodegeneration that might help to unravel the etiologies of diseases and, in the future, prompt the development of new therapies to prevent them; however, more studies are required. These studies should be directed toward using similar tissue sampling and gene expression and confirmed by quantitative RT-PCR as well as proteomic profiling. One inherent problem with gene expression studies is that not all changes in gene expression can always be correlated with specific proteins. Conflict of interest The authors have declared no conflicts of interest. Acknowledgements We wish to thank the National Parkinson Foundation (Miami, USA), the Michael J. Fox Foundation (New York, USA), the Bayerische Julius-Maximilians University of Wu¨rzburg

and Technion Research and Development for their generous support of this work. References [1] Gru¨nblatt E. The benefits of microarrays as a tool for studying neuropsychiatric disorders. Drugs Today 2004;40(2):147e56. [2] Mandel S, Weinreb O, Youdim MB. Using cDNA microarray to assess Parkinson’s disease models and the effects of neuroprotective drugs. Trends Pharmacol Sci 2003;24:184e91. [3] Tomita H, Vawter MP, Walsh DM, Evans SJ, Choudary PV, Li J, et al. Effect of agonal and postmortem factors on gene expression profile: quality control in microarray analyses of postmortem human brain. Biol Psychiatry 2004;55:346e52. [4] Hynd MR, Lewohl JM, Scott HL, Dodd PR. Biochemical and molecular studies using human autopsy brain tissue. J Neurochem 2003;85:543e62. [5] Hauser MA, Li YJ, Takeuchi S, Walters R, Noureddine M, Maready M, et al. Genomic convergence: identifying candidate genes for Parkinson’s disease by combining serial analysis of gene expression and genetic linkage. Hum Mol Genet 2003;12:671e7. [6] Gru¨nblatt E, Mandel S, Jacob-Hirsch J, Zeligson S, Amariglo N, Rechavi G, et al. Gene expression profiling of parkinsonian substantia nigra pars compacta; alterations in ubiquitineproteasome, heat shock protein, iron and oxidative stress regulated proteins, cell adhesion/cellular matrix and vesicle trafficking genes. J Neural Transm 2004;111: 1543e73. [7] Lu L, Neff F, Dun Z, Hemmer B, Oertel WH, Schlegel J, et al. Gene expression profiles derived from single cells in human postmortem brain. Brain Res Brain Res Protoc 2004;13:18e25. [8] Lu L, Neff F, Alvarez-Fischer D, Henze C, Xie Y, Oertel WH, et al. Gene expression profiling of Lewy body-bearing neurons in Parkinson’s disease. Exp Neurol 2005;195:27e39. [9] Lu L, Neff F, Fischer DA, Henze C, Hirsch EC, Oertel WH, et al. Regional vulnerability of mesencephalic dopaminergic neurons prone to degenerate in Parkinson’s disease: a post-mortem study in human control subjects. Neurobiol Dis 2006;23:409e21. [10] Chung CY, Seo H, Sonntag KC, Brooks A, Lin L, Isacson O. Cell typespecific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection. Hum Mol Genet 2005;14:1709e25. [11] Zhang Y, James M, Middleton FA, Davis RL. Transcriptional analysis of multiple brain regions in Parkinson’s disease supports the involvement of specific protein processing, energy metabolism, and signaling pathways, and suggests novel disease mechanisms. Am J Med Genet B Neuropsychiatr Genet 2005;137:5e16. [12] Hauser MA, Li YJ, Xu H, Noureddine MA, Shao YS, Gullans SR, et al. Expression profiling of substantia nigra in Parkinson disease, progressive supranuclear palsy, and frontotemporal dementia with parkinsonism. Arch Neurol 2005;62:917e21. [13] Papapetropoulos S, Shehadeh L, McCorquodale D. Optimizing human post-mortem brain tissue gene expression profiling in Parkinson’s disease and other neurodegenerative disorders: from target ‘‘fishing’’ to translational breakthroughs. J Neurosci Res 2007;85(14):3013e24.

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