Expression Profiling of Substantia Nigra in Parkinson Disease, Progressive Supranuclear Palsy, and Frontotemporal Dementia with Parkinsonism

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 parkinsonism 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 supranuclear 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 information 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-

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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

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©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 −1.5 .01 −1.0 .9 202500_at DNAJB2 −1.5 .03 −1.6 .07 Ubiquitination 200964_at UBE1 −1.4 .006 −1.2 .2 201387_s_at PARK5 −2.0 .01 −1.1 .7 Mitochondrial 200657_at SLC25A5 −1.4 .003 −1.1 .8 213735_s_at COX5B −1.3 .003 −1.1 .4 201754_at COX6C −1.3 .005 1.1 .6 218563_at NDUFA3 −1.3 .006 −1.2 .1 209492_x_at ATP5I −1.4 .006 1.0 .7 200697_at HK1 −1.4 .007 1.2 .5 202698_x_at COX4I1 −1.3 .01 −1.3 .04 201243_s_at ATP1B1 −1.5 .01 1.4 .2 Vacuolar 201441_at COX6B −1.4 .01 −1.3 .2 200030_s_at SLC25A3 −1.4 .01 1.1 .7 201757_at NDUFS5 −1.4 .02 1.1 .5 200818_at ATP5O −1.4 .02 −1.1 .8 202233_s_at UQCRH −1.4 .02 1.1 .8 208870_x_at ATP5C1 −1.3 .03 1.1 .6 201226_at NDUFB8 −1.7 .03 −1.1 .8 207507_s_at ATP5G3 −1.5 .03 1.0 .9 205711_x_at ATP5C1 −1.2 .04 1.2 .3 208845_at VDAC3 −1.3 .04 1.0 .8 200086_s_at COX4I1 −1.3 .04 −1.3 .1 202110_at COX7B −1.3 .04 −1.2 .4 206790_s_at NDUFA9 −1.4 .04 1.0 .7 201242_s_at ATP1B1 −1.5 .047 1.2 .04 212383_at ATP6V0A1 −1.4 Ͻ.001 −1.1 .5 212041_at ATP6V0D1 −1.6 .001 1.1 .6 36994_at ATP6V0C −1.6 .003 −1.0 .9 201089_at ATP6V1B2 −2.0 .007 −1.0 .9 203909_at SLC9A6 −1.4 .01 −1.0 .8 207809_s_at ATP61P1 −1.4 .02 1.2 .2 208898_at ATP6V1D −1.3 .03 −1.0 .9 200078_s_at ATP6V0B −1.3 .04 1.1 .5 201972_at ATP6V1A1 −1.5 .04 1.0 .9 208678_at ATP6V1E1 −1.3 .045 −1.1 .3 Vesicle trafficking 201864_at GDI1 −1.3 .009 −1.1 .1 202260_s_at STXBP1 −1.7 .02 −1.1 .8 203999_at SYT1 −2.6 .03 −2.0 .3 213326_at VAMP1 −2.1 .045 1.1 .8

Abbreviations: FTDP, frontotemporal dementia with parkinsonism; ID, identifier; ND, not detected above threshold expression level; PSP, progressive supranuclear palsy.

the corresponding author). These are potential PD sus- COMMENT ceptibility genes, as they are functional (expression) and positional (linkage) candidates. Finally, we explored the potential to use gene expres- Our global expression profiling of neural tissue from PD, sion to place the samples into diagnostic groups. We used PSP, and FTDP patients identified 130 prioritized can- 226 genes (142 PD vs control, and 84 PSPϩFTDP vs con- didate genes, correlating well with expression stud- trol) (Figure, A) to perform supervised hierarchical clus- ies2,3,14 of model systems for PD (Table 3). It also iden- tering. Although this was unsuccessful (Figure, B), after tified 12 genes that may reflect secondary changes due removing the 12 secondary effect genes, the samples fell to neuronal loss. Despite the limited sample size, re- into 3 distinct clusters, with only a single PD sample that moval of these genes increased the specificity of super- was misclassified (Figure, C). vised hierarchical clustering, suggesting that a formal clas-

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 A Table 3. Expression Changes in Parkinson Disease Model Systems 130 12 84 Human Drosophila Drosophila PD vs Control PSP + FTDP vs Control Molecular Substantia R406W A30P Pathway Nigra MAPT2 ␣-Synuclein2 Yeast3 Chaperones ϩϩϩ ϩϩϩ ϩ ϩϩ ϩϩϩ ϩϩ B Ubiquitination ...... Vesicle trafficking ϩϩϩ ϩϩ ϩϩ ϩϩϩ Mitochondrial ϩϩϩ ... ϩϩϩ ϩ Apoptosis ϩϩϩ ......

Abbreviations: MAPT, microtubule-associated protein tau gene; ϩ, slightly involved; ϩϩ, moderately involved; ϩϩϩ, very involved.

ous observation of decreases in complex I and complex IV activity in PD.18 This is unlikely to be secondary to reduced metabolic activity resulting from neuronal death: PD872 PD258 PD185 PD81 PSP461 CONTROL543 CONTROL673 PD214 PD154 CONTROL1037 CONTROL911 CONTROL1035 FTDP718 PSP742 only 2 (COX4I1 and ATP1B1) of these 22 genes are also significantly reduced in PSP and FTDP, while 13 are elevated. This supports the recently postulated model of C complex I dysfunction being the central player in initi- ating PD.18 The ␣-synuclein fly shows similar reduc- tions in energy metabolism genes at early presymptom- atic time points,2 although this trend is reversed later in the course of disease. Intriguingly, PD (but not PSP or FTDP) patients ex- press decreased levels of transcripts involved in protein trafficking, in general, and in neurotransmitter secre- tion, in particular. Vacuolar adenosine triphosphatases are involved in protein sorting and receptor-mediated en- docytosis and have been directly implicated in neuro- FTDP718 PSP742 PSP461 PD81 PD185 PD872 PD258 PD154 PD214 CONTROL543 CONTROL673 CONTROL1037 CONTROL911 CONTROL1035 transmitter release.19 Eight different subunits of vacu- Figure. Cluster analysis. A, Venn diagram of genes used in cluster analysis. olar adenosine triphosphatase are significantly B, Supervised hierarchical cluster analysis with 238 genes (142 Parkinson underexpressed in PD SN compared with control speci- disease [PD] vs control and 96 progressive supranuclear palsy and mens, correlating with the reduced expression of a novel frontotemporal dementia with parkinsonism [PSPϩFTDP] vs control). C, The same analysis excluding the 12 secondary effect genes. lysosomal hydrogen adenosine triphosphatase seen in the ␣-synuclein fly.2 Neuronal exocytosis requires docking of multiple membrane proteins, such as synaptobrevin, sification analysis with more samples and appropriate which was reduced by more than 2-fold in PD SN. Even cross-validation may be able to distinguish PD from PSP this small change could be biologically important, as syn- and FTDP. aptobrevin is normally present in stoichiometrically lim- We demonstrate increases in heat shock proteins iting amounts.20 The protein STXBP1 binds to syntaxin HSPA1A and HSPA1B in PD, PSP, and FTDP compared on the target membrane, forming part of the parallel 4-he- with control SN, indicating that this may be a common lix bundle that is thought to drive the fusion of oppos- response to mitigate the toxic effects of misfolded pro- ing membranes.20 After membrane docking, calcium binds tein. This is supported by the ability of Hsp70 to reverse to synaptotagmin, triggering neurotransmitter release at the phenotype of the ␣-synuclein transgenic fly15 and by the synapse.21 Our microarray analysis showed that ex- the up-regulation of endogenous chaperones in R406W pression levels of STXBP1 and synaptotagmin are sig- microtubule-associated protein tau (MAPT) flies.2 nificantly reduced in PD SN. This pathway is implicated Mutations in the ubiquitination genes UCHL1 in the Drosophila and yeast PD models: the A30P fly shows (PARK5)16 and parkin (PARK2)17 have previously been abnormal expression levels of lipid genes and the reti- found in patients with PD. Our analysis shows that PARK5 noid and fatty acid–binding glycoprotein gene (RFABG),2 is reduced 2-fold in PD. Variants of this protein have been and 18 of 57 genes implicated in the yeast PD model were associated with increases in ␣-synuclein levels in cul- clustered in the functionally related categories of lipid tured cells.16 The ubiquitin-activating enzyme E1 tran- metabolism and vesicle-mediated transport.3 script is also reduced in PD SN. These observations are Our microarray expression analysis of SN tissue from consistent with a general pattern of accumulation of ab- patients with PD identified candidate PD susceptibility normal protein in PD and are probably not secondary ef- genes and pathways, the importance of which is corrobo- fects; they were not detected in the PSP or FTDP samples. rated in PD model systems. We used expression analy- We find a decrease in expression of 22 nuclear- sis of the related neurodegenerative diseases PSP and encoded mitochondrial proteins, consistent with previ- FTDP to identify genes that may reflect secondary changes.

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 Finally, we identified expression differences between PD, cially, the patients and their families, whose generosity PSP, and FTDP that suggest a potential role for micro- and support made this research possible. array analysis in future postmortem diagnostic proce- dures. Further studies with increased sample sizes and laser capture microdissection should provide further in- sight into this potential. REFERENCES

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