ORIGINAL CONTRIBUTION Expression Profiling of Substantia Nigra in Parkinson Disease, Progressive Supranuclear Palsy, and Frontotemporal Dementia With Parkinsonism Michael A. Hauser, PhD; Yi-Ju Li, PhD; Hong Xu, MA; Maher A. Noureddine, PhD; Yujun S. Shao, PhD; Steven R. Gullans, PhD; Clemens R. Scherzer, MD; Roderick V. Jensen, PhD; Adam C. McLaurin, BA; Jason R. Gibson, BA; Burton L. Scott, MD; Rita M. Jewett, RN; Judith E. Stenger, PhD; Donald E. Schmechel, MD; Christine M. Hulette, MD, PhD; Jeffery M. Vance, MD, PhD Background: Parkinson disease (PD) is characterized Results: There were 142 genes that were significantly by loss of dopaminergic neurons in the substantia nigra. differentially expressed between PD cases and controls Genes contributing to rare mendelian forms of PD have and 96 genes that were significantly differentially ex- been identified, but the genes involved in the more com- pressed between the combined progressive supra- mon idiopathic PD are not well understood. nuclear palsy and frontotemporal dementia with parkin- sonism cases and controls. The 12 genes common to all Objectives: To identify genes important to PD patho- 3 disorders may be related to secondary effects. Hierar- genesis using microarrays and to investigate their poten- chical cluster analysis after exclusion of these 12 genes tial to aid in diagnosing parkinsonism. differentiated 4 of the 6 PD cases from progressive su- pranuclear palsy and frontotemporal dementia with par- Design: Microarray expression analysis of postmortem kinsonism. substantia nigra tissue. Conclusions: Four main molecular pathways are al- Patients: Substantia nigra samples from 14 unrelated tered in PD substantia nigra: chaperones, ubiquitina- individuals were analyzed, including 6 with PD, 2 with tion, vesicle trafficking, and nuclear-encoded mitochon- progressive supranuclear palsy, 1 with frontotemporal de- drial genes. These results correlate well with expression mentia with parkinsonism, and 5 control subjects. analyses performed in several PD animal models. Ex- pression analyses have promising potential to aid in post- Main Outcome Measures: Identification of genes sig- mortem diagnostic evaluation of parkinsonism. nificantly differentially expressed (PϽ.05) using Af- fymetrix U133A microarrays. Arch Neurol. 2005;62:917-921 ARKINSON DISEASE (PD) (ON- tients with progressive supranuclear palsy line Mendelian Inheritance in (PSP) (Online Mendelian Inheritance in Man 168600) is character- Man 601104) and frontotemporal demen- ized by progressive degenera- tia with parkinsonism (FTDP) (Online Men- tion of central pathways, from delian Inheritance in Man 600274), in ad- the dorsal motor nuclei, then to the hall- dition to patients with PD and control P 5 6 mark dopaminergic neurons of the substan- subjects. Patients with PSP and FTDP have tia nigra (SN), and finally to additional re- clinical presentations that are similar to gions such as the neocortex.1 Expression those of patients with PD and exhibit neu- analysis can support and extend these patho- ronal loss with gliosis in the SN. Profiling logic descriptions, provide new insights into of FTDP and PSP allows identification of ex- the disease process, and potentially aid in pression changes that may be due to changes diagnosis. It also facilitates the compari- in the cell populations of the SN associ- son of anatomically different Drosophila2 and ated with disease and should enrich our yeast3 PD models. Finally, these data can be knowledge of PD-specific changes. coupled with linkage and other genetic in- formation to identify risk and modifier genes METHODS for PD susceptibility.4 Expression studies identify changes that DIAGNOSTIC CRITERIA are specific to the disease, as well as down- stream secondary effects. To characterize Parkinson disease was diagnosed pathologi- Author Affiliations are listed at these secondary changes, we conducted mi- cally and staged according to the methods of the end of this article. croarray analysis on SN tissue from pa- Braak et al1 for PD and Alzheimer disease. Clini- (REPRINTED) ARCH NEUROL / VOL 62, JUNE 2005 WWW.ARCHNEUROL.COM 917 ©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 Table 1. Tissue Donor Information Sample No. Diagnosis Age, y Sex PD Braak Stage AD Braak Stage Postmortem Delay, h:min 673 Control 81 F NA NA 1:10 543 Control 72 F NA NA 3:00 1037 Control 88 F NA NA 20:30 1035 Control 72 F NA NA 30:00 911 Control 90 M NA NA 7:25 81 PD 87 M III II 2:30 214 PD 84 M IV II 2:45 872 PD 82 F IV I 15:00 258 PD 79 F VI II 12:45 185 PD 83 M III IV 00:42 154 PD 74 M V II 5:25 461 PSP 70 M NA NA 13:50 742 PSP 64 F NA NA 2:00 718 FTDP 58 F NA NA 24:00 Abbreviations: AD, Alzheimer disease; FTDP, frontotemporal dementia with parkinsonism; NA, not available; PD, Parkinson disease; PSP, progressive supranuclear palsy. cal records were reviewed by a movement specialist (B.L.S.) to erarchical clustering was performed using Cluster (http://rana ensure that the subjects met previously reported criteria.7 Lewy .lbl.gov/EisenSoftware.htm) with the complete linkage option body pathologic evidence was evaluated according to consen- and visualized using TreeView (http://rana.lbl.gov/EisenSoftware sus guidelines8 and PSP according to National Institute of Neu- .htm). Affymetrix hybridization probes were mapped to ge- rological Disorders and Stroke criteria.9 Frontotemporal de- nomic linkage peaks as previously described.4 mentia with parkinsonism linked to chromosome 17q21-22 has been described.6 All 9 patient samples showed typical patho- RESULTS logic features, including moderate to severe neuronal loss and gliosis. Control subjects were cognitively normal, died of non- neurological causes, and had no clinical or pathological evi- Affymetrix U133A chips were used to measure SN gene dence of a movement disorder. expression from 6 PD, 2 PSP, 1 FTDP, and 5 control samples. First, the 6 PD samples were compared with the PROCUREMENT OF RNA 5 control samples, revealing 142 (122 reduced and 20 elevated) significantly differentially expressed genes At autopsy, brain hemispheres were frozen in liquid nitrogen (PϽ.05) (a table containing this supplemental informa- and stored at −80°C in the Kathleen Price Bryan Brain Bank in tion is available from the corresponding author). Fold the Alzheimer’s Disease Research Center at Duke University.10 changes (Յ4-fold) are consistent with those seen in other Using the RNAgents kit (Promega, Madison, Wis), RNA was investigations.2 Table 2 gives a subset of these genes that extracted from SN and adjacent midbrain tissues. The delay be- fall into molecular pathways previously associated with fore postmortem examinations varied (Table 1); however, brain 11 PD. This differential expression has been confirmed us- messenger RNA is stable for up to 36 hours after death. Double- 12 stranded complementary DNA was made with a biotinylated ing serial analysis of gene expression. The 142 genes T7(dT)-24 primer. and others in the same pathways are candidates for PD susceptibility and phenotypic modifier genes, and will MICROARRAYS be tested by association analysis in patients with PD and controls.13 Twenty micrograms of biotinylated complementary RNA was The SN of patients with PD shows many secondary fragmented and hybridized to Affymetrix human genome U133A effects of disease (eg, neuronal loss and gliosis) that may microarrays (Affymetrix Inc, Santa Clara, Calif). Affymetrix Mi- induce expression changes unrelated to disease cause or croarray Suite 5.0 software was used for global scaling, with a progression. The PSP and FTDP samples analyzed also mean “target intensity” of 100 for all probe sets. To control for show loss of dopaminergic neurons and should exhibit partial RNA degradation, 3Ј/5Ј ratios for glyceraldehyde-3- the same secondary effects. We identified 96 genes that phosphate dehydrogenase probes were examined (M33197_5_at were significantly differentially expressed between PSP and M33197_3_at). Of 19 original samples, 5 (1 PD, 3 con- and FTDP cases and controls (PϽ.05) (a table contain- trol, and 1 PSP) with 3Ј/5Ј end ratios greater than 5.0 were ex- cluded from analysis. ing this supplemental information is available from the corresponding author). Twelve of these genes were also differentially expressed between PD and control SN DATA ANALYSIS (Figure, A). We hypothesize that these genes reflect sec- We analyzed 1164 probe sets with mean intensities of at least ondary effects common to all 3 disorders and should be given less priority in the search for genes involved in PD 500. After log2 transforming the raw intensities, differentially expressed genes were identified using a 2-sample t test. This pathogenesis, leaving 130 prioritized genes. Twenty of study was hypothesis generating rather than hypothesis test- these 130 genes map to regions of PD linkage7 (a table ing, so we report nominal P values with ␣=.05. Supervised hi- containing this supplemental information is available from (REPRINTED) ARCH NEUROL / VOL 62, JUNE 2005 WWW.ARCHNEUROL.COM 918 ©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 Table 2. Selected Genes Differentially Expressed Between Parkinson Disease (PD) and Control Substantia Nigra PD vs Control PSP؉FTDP vs Control Human Genome Microarray (Clone)_ID Organization Name Fold Change P Value Fold Change P Value Chaperones 202581_at HSPA1B 2.5 .008 ND ND 200799_at HSPA1A 2.6 .007 2.1 .005 204720_s_at DNAJC6