lable at ScienceDirect

Neurobiology of Aging 36 (2015) 1686e1691

Contents lists avai

Neurobiology of Aging

journal homepage: www.elsevier .com/locate/neuaging

Brief communication

Upregulation of alphaB-crystallin expression in the substantia nigraof patients with Parkinson’s disease

Yingjun Liu a, Qinbo Zhou a, Mi Tang a,b, Ning Fu c, Wei Shao a, Shuzhen Zhang a,Yanqing Yin a, Rong Zeng c, Xiaomin Wang d, Gang Hu b, Jiawei Zhou a,*

a Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science, Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences, Shanghai, Chinab Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu, ChinacKey Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,Shanghai, ChinadCenter of Parkinson’s Disease, Beijing Institute for Brain Disorders, Beijing, China

a r t i c l e i n f o

Article history:Received 21 September 2014Received in revised form 18 November 2014Accepted 16 January 2015Available online 22 January 2015

Keywords:Parkinson’s diseaseQuantitative proteomicsHumanAlphaB-crystallinGlial cells

* Corresponding author at: Institute of NeuroscienNeuroscience, Shanghai Institutes for Biological Scienences, 320 Yueyang Rd, Shanghai 200031, China. Tel./

E-mail address: jwzhou@ion.ac.cn (J. Zhou).

0197-4580/$ e see front matter � 2015 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.neurobiolaging.2015.01.015

a b s t r a c t

Parkinson’s disease (PD) is one of the most devastating neurodegenerative disorders. The underlyingmechanisms of the characteristic neurodegeneration in the substantia nigra (SN) are still not fully un-derstood. To better understand the molecular events occurring in the SN of PD brain, we used theculture-derived isotope tagebased quantitative proteomics to compare the protein expression profiles inthe nigral tissue of PD patients and control subjects. We identified a total of 11 differentially expressedproteins, including alphaB-crystallin (Cryab). Both the levels and pattern of Cryab expression in the SNwere validated. It was revealed that Cryab was markedly upregulated in the SN of PD brain. Cryabexpression was also upregulated in reactive astrocytes and microglia in a neurotoxin-induced mouse PDmodel. Moreover, we showed increased expression of Cryab in cytoplasmic inclusions in a subset of glialcells in Parkinsonian brain. Thus, we identified Cryab that is highly expressed in the SN of PD brain andmay be involved in the glial pathology during dopaminergic neuron degeneration in PD.

� 2015 Elsevier Inc. All rights reserved.

1. Introduction

Parkinson’s disease (PD) is a common neurodegenerativedisorders affecting millions of people around the world. It is char-acterized by selective degeneration of dopaminergic (DA) neuronsin the substantia nigra (SN). Patients with PD are afflicted by severemotor symptoms including resting tremor, bradykinesia, rigidity,and postural instability. After decades of intensive investigation,several hypotheses regarding the causes and pathogenesis of PDhave been proposed, such as oxidative stress, mitochondrialdysfunction, protein aggregation, and inflammation (Foltynie andKahan, 2013; Jenner, 1991; McGeer et al., 2001; McNaught andOlanow, 2006; Mizuno et al., 1998). However, no curative thera-peutics for PD has been successfully developed until now based onthe current understanding of the disease, highlighting the impor-tance of identifying new therapeutic targets.

ce, State Key Laboratory ofces, Chinese Academy of Sci-fax: þ86 21 5492 1073.

ll rights reserved.

The application of high-throughput approaches, such as DNAmicroarray and proteomics, has dramatically enhanced our under-standing of the pathophysiology of several neurologic diseasesincluding PD. DNA microarray analysis evaluates gene expressionon a genome scale by using brain tissues from animal models orhuman patients. By using this powerful method, a wide variety ofgenes, including PGC-1a, that is linked to pathogenic changes in PDhas been identified (Mandel et al., 2005; Simunovic et al., 2009;Zheng et al., 2010). However, given the discrepancy between themessenger RNA and protein abundance in the biological samples,proteomics has the advantage of allowing direct comparison of thechanges in protein levels among various conditions.

One of the drawbacks of traditional proteomic approaches isthe inability to make quantitative comparison on the absolute orrelative protein levels across biological samples. Recent advancesin the development of quantitative methods in proteomics pro-vide unprecedented opportunity to quantify protein levels incomplex biological samples. One of such methods is based on theuse of culture-derived isotope tags (CDITs), in which the isotope-labeled proteins from cell culture were introduced as internalstandards to quantify the protein levels in complex tissue samples

Y. Liu et al. / Neurobiology of Aging 36 (2015) 1686e1691 1687

(Ishihama et al., 2005). Here, we quantitatively compared theproteomes of the SN from PD patients and healthy controls byusing CDIT method. We found 11 proteins that displayed>1.5-fold change between control and disease groups. Among the11 proteins, the upregulation of alphaB-crystallin (Cryab) wasvalidated by using western blot in human brain samples. Immu-nohistochemistry revealed aberrant expression patterns of Cryabin the SN of PD patients and neurotoxin-induced rodent PDmodel. Our study provides compelling evidence that Cryab isinvolved in PD pathogenesis.

2. Materials and methods

2.1. Human tissue collection

Fresh-frozen ventral mesencephalic tissues and tissue sectionswere obtained from The Netherlands Brain Bank, NetherlandsInstitute for Neuroscience, Amsterdam, The Netherlands. All thematerials have been collected from donors for or from whom awritten informed consent for a brain autopsy and the use of thematerial and clinical information for research purposes had beenobtained by The Netherlands Brain Bank. For proteomic and posthoc biochemical analyses, frozen brain tissues from 3 pairs of age-and gender-matched PD patients and healthy controls(Supplementary Table S1) were used. After 3 washes by coldphosphate-buffered saline, brain tissues were homogenized in 50-mM Tris (pH 7.4), 150-mM NaCl, 1% NP-40, 0.1% sodium dodecylsulfate, and 1% sodium deoxycholate. A set of paraffin-embeddedsections from 4 PD and 3 healthy control subjects were obtainedfrom the same source and were used for immunohistochemicalanalysis. All the PD subjects were clinically and neuropathologicallydiagnosed, and the healthy control subjects were devoid of anyneurologic diseases.

2.2. Quantitative proteomic analysis

Quantitative proteomic analysis was carried out as describedpreviously (Ishihama et al., 2005) with a few modifications. Briefly,human SH-SY5Y neuroblastoma cells were grown in Dulbecco-modified Eagle medium deficient in L-leucine (Sigma-Aldrich, StLouis, MO, USA) with 10% fetal bovine serum (Gibco, NY, USA), and13C-labeled L-leucine (Bio-Rad, Hercules, CA, USA) was added to theculture. After 6 passages, proteins were extracted and mixed withhuman brain samples. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins were recovered andanalyzed by liquid chromatography in combination with tandemmass spectrometry (MS) (Thermo, San Jose, CA, USA). After proteinidentification by MS, leucine-containing peptides were extracted.Their m/z and scan number information were used to extract masschromatograms and the peak areas of peptides, and manualconfirmation was done to correct peak areas. Each peak wasquantified relative to its corresponding isotope-labeled peak fromSH-SY5Y cells, which were used as comprehensive internal stan-dards to normalize the variations of sample preparation and anal-ysis. Finally, the amount of each peak was compared in differenttissue samples relative to SH-SY5Y cells.

2.3. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model ofPD

Adult male C57BL/6 mice obtained from Shanghai LaboratoryAnimal Center, Chinese Academy of Sciences, were maintained on a12-hour light-dark cycle at 23 �Cwith food andwater ad libitum. Allthe procedures were approved by the Institutional Animal Care andUse Committee and were in accordance with the US National

Institutes of Health Guide for the Care and Use of Laboratory Ani-mals. The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)einduced mouse model of PD was prepared as described previously(Jackson-Lewis and Przedborski, 2007). Briefly, the mice (8- to 10-week olds) received 4 injections (with 2-hour intervals) of MPTP(20 mg/kg, intraperitoneal; Sigma-Aldrich) or saline in 1 day. Sevendays after the last injection, mice were anesthetized by pentobar-bital sodium and perfused transcardially with saline, followed by 4%paraformaldehyde. Brains were collected and postfixed until use.

2.4. Western blot analysis

Western blotting was performed as described previously (Liet al., 2006). The primary antibodies used were mouse mono-clonal antibody against b-actin (1:5000, Sigma-Aldrich) andmonoclonal antibody against Cryab (1:1000, Santa Cruz Biotech-nology, Santa Cruz, CA, USA). The membranes were washed andincubated with appropriate secondary antibodies (1:5000, JacksonImmunoResearch Laboratories, West Grove, PA, USA). Membraneswere visualized and digitized with ImageQuant (LAS-4000; Fuji-film, Tokyo, Japan). Optical densities of bands were analyzed byusing ImageReader software (Fujifilm).

2.5. Immunohistochemistry

Immunostaining on paraffin-embedded sections of humanmidbrain were performed. The sections were treated to retrieveantigens followed by incubation with 0.3% H2O2 in phosphate-buffered saline for 30 minutes to bleach the endogenous peroxi-dase. Sections were incubated with primary mouse anti-Cryabmonoclonal antibody (1:200, Santa Cruz Biotechnology) followedby biotinylated secondary antibodies (1:800; Jackson ImmunoR-esearch Laboratories). Immunosignals were visualized by using 3,3-diaminobenzidine (Sigma-Aldrich), and images were captured by amicroscope (BX51; Olympus, Tokyo, Japan). For mouse brain sec-tions (25 mm thickness), similar protocols were used. For doubleimmunofluorescent staining, mouse brain sections were incubatedwith either polyclonal rabbit anti-glial fibrillary acidic protein(GFAP, 1:1000; Dako, Denmark) or ionized calcium-binding adaptermolecule 1 (Iba1, 1:500; Wako, Wakayama, Japan) antibody fol-lowed by incubation with secondary antibody conjugated withAlexa488. The same sections were then incubated with monoclonalCryab antibody (1:200; Santa Cruz Biotechnology), followed byincubation with secondary antibody conjugated with Alexa555.Images were captured and analyzed by using a laser confocal mi-croscope (Nikon, Tokyo, Japan).

2.6. Statistical analysis

Statistical analysis was performed using statistical software(GraphPad Prism, version 4.0; GraphPad Software, Inc, La Jolla, CA,USA). The control group was compared with the PD group usingStudent t test. Differences were considered significant when pvalues were <0.05.

3. Results

To investigate the molecular events occurring in PD brain, wequantitatively compared the proteomes of the nigral tissue from the3 patients with PD and 3 age- and gender-matched healthy controlsubjects, using the CDIT-based quantitative proteomic approach.The human DA SH-SY5Y cells were cultured in 13C-labeled leucine-rich medium and then mixed with human brain samples fromeither PD or healthy controls to serve as an internal standard. A totalof 3934 proteins were identified by MS, among which 229 proteins

Y. Liu et al. / Neurobiology of Aging 36 (2015) 1686e16911688

were presented in all the samples. To evaluate the coverage of the229 proteins in terms of their cellular and functional distributions,we analyzed these proteins by NetAffx software (Liu et al., 2003). Itwas revealed that the cellular and functional distributions of theseproteins were highly divergent, indicating good coverage of themass spectrometry readouts (Supplementary Fig. S1A and B).

We then assigned proteins with >1.5-fold changes in all the 3pairs of brain samples as differentially regulated proteins in PD. Atotal of 11 such proteins were identified, 6 of which, includingCryab, hyaluronan and proteoglycan link protein 2 (Hapln2), coiled-coil domain containing 94 (Ccdc94), membrane-associated pro-gesterone receptor component 1 (Pgrmc1), heterogeneous nuclearribonucleoprotein Uelike 2 (Hnrnpul2), and proteasome activatorcomplex subunit 2 (Psme2), were upregulated in the SN of patientswith PD compared with control subjects. The 5 proteins that weredownregulated in PD samples were DUT protein (Dut), potassiumchannel tetramerization domainecontaining protein 13 (Kctd13),tubulin alpha-1 chain (Tba1), ruvB-like AAA ATPase 1 (Ruvb1), andannexin a1 (Anxa1) (Supplementary Table S2). Considering that thesamples used to generate this data set were from patients at late-stage PD when >50%e70% of DA neurons had already degen-erated, the downregulation of some of these proteins may partiallyresult from DA neuron loss.

To validate whether the expression levels of the differentiallyexpressed proteins were truly altered, Cryab was selected forfurther analysis. It was shown that expression levels of Cryab pro-tein were markedly increased in the nigral tissue of patients withPD compared with control subjects (Fig. 1A and B). Consistent with

Fig. 1. AlphaB-crystallin (Cryab) is upregulated in the substantia nigra (SN) of patients withthe SN of PD patients and healthy controls. (B) Quantitative data shown in (A). Data are expreelevated and aggregated in glial cells in the SN of PD. Immunohistochemical staining for CEnlarged images of the squared areas in (C) and (D), respectively. Cryab expression in most oflevel of Cryab is higher, showing intense immunosignals in both of the cell body and cell extCryab expression is significantly elevated, and the protein aggregates in a fraction of glial celland (I), 20 mm; and (J) and (K), 8 mm.

these data, MPTP administration in mice also elicited robust in-creases in Cryab expression. Immnunohistochemical analysisrevealed that in the SN of MPTP-treated mice, there was a dramaticincrease of Cryab expression in the SN, as evidenced by thedramatically elevated intensity of Cryab immunosignals in the SN ofMPTP mouse model of PD compared with the control(Supplementary Fig. S2A). Notably, aberrant Cryab expressionoccurred in reactive glial cells, indicating that Cryab-positive glialcells represent the classical glial scar in the SN of MPTP mouse PDmodel. Moreover, double immunofluorescent staining showed thatCryab was expressed in reactive astrocytes (SupplementaryFig. S2B), as manifested by colocalization of Cryab and GFAP inthe SN of MPTP-treated mice. Interestingly, colocalization of Cryaband Iba1, a marker for microglial cells, was also observed in the SNof MPTP-treated mice (Supplementary Fig. S2C), whereas restingmicroglial cells in control subjects were devoid of Cryab immuno-signals (data not shown). Taken together, these results suggest thatCryab is associated with gliosis in a mouse PD model. ElevatedCryab may be neuroprotective by preventing neuronal degenera-tion induced by pathogenic stimuli.

To determine the cellular basis of Cryab upregulation in humanbrain, we investigated the expression pattern of Cryab in the SN ofan independent cohort of PD patients and healthy controls by usingimmunohistochemistry. These PD patients had been pathologicallydiagnosed by the presence of alpha-synucleinepositive intra-cytoplasmic inclusions (Lewy body), in addition to the clinicaldiagnosis. Indeed, the alpha-synucleinecontaining intra-cytoplasmic inclusions were observed in the SN of all 4 subjects

Parkinson’s disease (PD). (A) Representative western blots showing Cryab expression inssed as mean � standard error of the mean (n ¼ 3), *p < 0.05. (CeK) Cryab expression isryab in the SN of the subjects with PD (GeK) or controls (CeF) are shown. (E) and (F)the cells in the control subjects is relatively low. However, in some cells, the expressionensions (arrows). (HeI) Enlarged images of the squared areas shown in (G). In PD brain,s in PD are Cryab positive (arrowheads). Scale bars: (C), (D), and (G), 50 mm; (E), (F), (H),

Y. Liu et al. / Neurobiology of Aging 36 (2015) 1686e1691 1689

with PD, but not in the 3 controls in our immunohistochemicalstaining (data not shown). Cryab immunohistochemical stainingrevealed that Cryabwas primarily localized in glial cells (Fig. 1C andD). In the brain of healthy subjects, the expression of Cryab in mostof the cells was relatively low, although the expression levels of glialCryab varied from one cell to another, and some of the Cryab-positive cells displayed higher intensity of immunoreactivities(Fig. 1CeF). In contrast, in the PD brain, the immunosignals of Cryabappeared to be significantly elevated compared with the controls(Fig. 1G). Interestingly, the increased expression of Cryab wasobserved in intracytoplasmic inclusions in glial cells but not inneuronal cells. Cryab was also observed in the neuropil thread pa-thology in cellular processes (Fig. 1HeK). In a few cases, cells withCryab-positive inclusion were dismantled (Fig. 1K), suggesting thatthey were dying cells during neurodegeneration.

4. Discussion

In the present study, we investigated the proteome profiles of PDby applying CDIT-based quantitative proteomics approach. Elevendifferentially expressed proteins were identified in the SN of PD,setting the basis for further investigation. The dysregulated pro-teins in PD are associated mainly with cell survival, proteindegradation, and neuroinflammation, supporting leading hypoth-esis on PD pathogenesis.

CDIT-based quantitative proteomics uses isotope-labeled pro-teins from cell culture as internal standards to quantify the sameproteins in the complex tissue samples. In the practice, usually only1 or 2 cell lines are used to generate isotope-labeled proteins thatare unlikely to cover all the proteins expressed in tissue samples.Therefore, only those proteins that are expressed in both the cellline(s) and tissues have a good chance of getting quantitated.Despite the limitations, this approach is still considered verypowerful, because of its quantitative nature, comparing withtraditional proteomic approaches that generate descriptive datathat are difficult to validate.

Among the upregulated proteins identified by our proteomicanalysis, Hapln2 had the highest fold change. This protein is knownto be localized in the extracellular matrix of the white matter in theadult central nervous system (CNS) and predominantly expressedby neurons (Hirakawa et al., 2000; Oohashi et al., 2002), functioningin the diffusion barrier formation and conduction velocity in theCNS (Bekku et al., 2010). However, the exact roles of Hapln2 in DAneurons and how it involves in PD pathogenesis are totally un-known. Pgrmc1 is a membrane-associated component of proges-terone receptor and expressed by neurons, astrocyte, andmicroglia.Interestingly, Pgrmc1 is upregulated in neurons and astrocyte aftertraumatic brain injury and in microglia when stimulated by lipo-polysaccharide or other inflammatory insults (Bali et al., 2013a,2013b; Meffre et al., 2005). Psme2 is one of the key regulators inthe proteasome activator PA28 complex. The upregulation of Psme2in PD brain may reflect the cellular responses to aggregated pro-teins such as alpha-synuclein, which is a key player in the patho-genesis of PD in both the familiar and sporadic cases. Importantly,Psme2 is also involved in the cellular immune responses by regu-lating the processing of major histocompatibility complex class Imolecules (Kloetzel et al., 1999; Sijts et al., 2002).

We found that 5 proteins, including Anxa1, Dut, Kctd13, Tuba4a,and Ruvb1, were downregulated in the brain of patients with PD.Previous comparative study of annexins in human brain foundintense cytoplasmic Anxa1 immunoreactivty in neurons, althoughit may also expressed in reactive astrocytes andmicroglia (Eberhardet al., 1994; McArthur et al., 2010). Interestingly, some of thedownregulated proteins in PD are important regulators for cellsurvival. Dut is an enzyme for deoxyuridine triphosphate hydrolysis

and a key regulator of intracellular deoxyuridine triphosphate poolsin the cell. Mitochondrial Dut is constitutively expressed and canprevent apoptotic cell death induced by exogenous and endogenousstresses (Williams et al., 2011). Ruvbl1 is an ATPase with diversebiological functions including, together with Ruvbl2, chromatinremodeling, transcription regulation, mitosis, and assembly oftelomere complex (Bauer et al., 2000; Ducat et al., 2008; Jonssonet al., 2001; Venteicher et al., 2008; Wood et al., 2000). Furtherstudies are required to investigate the precise function of thesedifferentially expressed proteins in PD pathogenesis.

Of the differentially expressed proteins identified in the SN inthis study, Cryab, which is expressed mainly in astrocytes and oli-godendrocytes in the adult brain, is known to be associated with avariety of pathologic conditions. Cryab is a member of small heat-shock protein family that actively responds to various cellularstresses such as aberrant protein aggregation under pathologicconditions and confers enhanced stress resistance on cells. Previousstudies showed that Cryab was involved in multiple neurologicdisorders such as tauopathies, Alexander’s disease, amyotrophiclateral sclerosis, PD, PD dementia, and prion disorders (Braak et al.,2001; Dabir et al., 2004; Head et al., 1993; Iwaki et al., 1989, 1992;Renkawek et al., 1999; Shinohara et al., 1993; Wang et al., 2013).These observations strongly suggest that Cryab may play animportant role in the pathogenesis of age-related CNS diseases.They also support the notion that PD share common pathogenicmechanism(s) to some extent with other neurodegenerative dis-eases and these diseases could potentially be targeted by similartherapeutic strategies. Interestingly, the expression pattern of Cryabin PD revealed in the present study is distinct from those reportedby others (Braak et al., 2001; Renkawek et al., 1999). In their studies,few of GFAPþ astrocytes reacted with Cryab antiserum were seen(Braak et al., 2001; Renkawek et al., 1999), despite that Cryab-positive neurons were evidenced in the cerebral cortex, amygdala,and hippocampus in PD brain (Braak et al., 2001). Moreover, Cryab-positive astrocytes can only be identified in PD dementia, a type ofdementia that develops in a patient who is originally diagnosedwith Parkinson’s, but not in those with typical PD (Renkawek et al.,1999), suggesting a strong correlation between aberrant Cryabexpression and cognitive impairments in these patients (Renkaweket al., 1999). In contrast, in the present study, our investigation onCryab expression in the SN of patients with PD using proteomicapproach in combination with classic biochemistry and histologyprovided compelling evidence that Cryab was elevated in glial cells,but not in neuronal cells, in the SN of patients with PD. Thediscrepancy in Cryab expression pattern between our observationsand those described by others may be attributable to the differentCryab antiserum used.

One of the remarkable features of the gliosis in PD presentedhere is the formation of abnormal Cryab-immunoreactive cyto-plasmic aggregation in glial cells in the SN of PD brain, which ap-pears to be similar in morphology to the glial inclusions of bothsporadic and familial tauopathies (Dabir et al., 2004). The impact ofglial inclusions on glial cell function is not yet clear. We speculatethat in the early stage of PD, Cryab may be upregulated in reactiveglial cells in response to inflammatory insults, such as MPTP asshown in Supplementary Fig. S2, and functions as a neuroprotectivemolecule, given that Cryab is known as a key negative regulator ofneuroinflammation (Masilamoni et al., 2005a, 2005b; Shao et al.,2013). As the disease progresses, detrimental factors induce theformation of glial inclusions via the sequestration of cytoplasmicproteins including Cryab through a yet-unidentifiedmode of action,thus tip the balance of immune homeostasis toward neuro-inflammation. Indeed, recent work in our laboratory has shownthat dramatic reduction in the levels of free Cryab protein in as-trocytes, induced by downregulation of astrocytic dopamine D2

Y. Liu et al. / Neurobiology of Aging 36 (2015) 1686e16911690

receptor that occurs in aging brain and PD, significantly promotesproinflammatory gene expression (Shao et al., 2013). The aggrega-tion of Cryab causes the impairment in Cryab-mediated proteinhandling and clearance promoting the recruitment of other pro-teins into aggregates in reactive glial cells and ultimately contrib-utes to the dysfunction of glial cells (Fig. 1J and K) andneurodegeneration.

In conclusion, the present study showed that Cryab wasmarkedly upregulated in the SN of PD patients where Cryab waspresent in glial cell inclusions. Further research is needed todetermine whether Cryab-containing inclusion is formed in earlystage of PD and exactly how Cryab is sequestered into the glialinclusion. It is of interest to note that the relevance between mostof the differentially expressed proteins identified in our proteomicanalysis is largely unexplored. Future studies are also required tofurther study the potential roles of these proteins in PD patho-genesis. The information generated from these studies will notonly promote our understanding to the PD pathogenesis but alsoprovide new insights into pathogenesis of neurodegenerativedisorders as a whole.

Disclosure statement

The authors have no conflicts of interest to disclose.

Acknowledgements

We thank the Netherlands Brain Bank for providing the humanbrain samples. We also thank J.N. Zhou for the help with brain tissuesamples. This work was supported by the grants from the ChineseAcademyofSciences, theNationalKeyBasicResearchProgramofChina(2011CB504102), the Natural Science Foundation of China (31123002,31430036, and 31321091), Shanghai Talent Award (Y45BN11241) andBeijing Institute for Brain Disorders (PXM2013_014226_07_000084).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.neurobiolaging.2015.01.015.

References

Bali, N., Arimoto, J.M., Morgan, T.E., Finch, C.E., 2013a. Progesterone antagonism ofneurite outgrowth depends on microglial activation via Pgrmc1/S2R. Endocri-nology 154, 2468e2480.

Bali, N., Morgan, T.E., Finch, C.E., 2013b. Pgrmc1: new roles in the microglialmediation of progesterone-antagonism of estradiol-dependent neurite sprout-ing and in microglial activation. Front. Neurosci. 7, 157.

Bauer, A., Chauvet, S., Huber, O., Usseglio, F., Rothbacher, U., Aragnol, D., Kemler, R.,Pradel, J., 2000. Pontin52 and reptin52 function as antagonistic regulators ofbeta-catenin signalling activity. EMBO J. 19, 6121e6130.

Bekku, Y., Vargova, L., Goto, Y., Vorisek, I., Dmytrenko, L., Narasaki, M., Ohtsuka, A.,Fassler, R., Ninomiya, Y., Sykova, E., Oohashi, T., 2010. Bral1: its role in diffusionbarrier formation and conduction velocity in the CNS. J. Neurosci. 30,3113e3123.

Braak, H., Del Tredici, K., Sandmann-Kiel, D., Rub, U., Schultz, C., 2001. Nerve cellsexpressing heat-shock proteins in Parkinson’s disease. Acta Neuropathol. 102,449e454.

Dabir, D.V., Trojanowski, J.Q., Richter-Landsberg, C., Lee, V.M., Forman, M.S., 2004.Expression of the small heat-shock protein alphaB-crystallin in tauopathieswith glial pathology. Am. J. Pathol. 164, 155e166.

Ducat, D., Kawaguchi, S., Liu, H., Yates 3rd, J.R., Zheng, Y., 2008. Regulation ofmicrotubule assembly and organization in mitosis by the AAAþ ATPase Pontin.Mol. Biol. Cell 19, 3097e3110.

Eberhard, D.A., Brown, M.D., VandenBerg, S.R., 1994. Alterations of annexinexpression in pathological neuronal and glial reactions. Immunohistochemicallocalization of annexins I, II (p36 and p11 subunits), IV, and VI in the humanhippocampus. Am. J. Pathol. 145, 640e649.

Foltynie, T., Kahan, J., 2013. Parkinson’s disease: an update on pathogenesis andtreatment. J. Neurol. 260, 1433e1440.

Head, M.W., Corbin, E., Goldman, J.E., 1993. Overexpression and abnormal modifi-cation of the stress proteins alpha B-crystallin and HSP27 in Alexander disease.Am. J. Pathol. 143, 1743e1753.

Hirakawa, S., Oohashi, T., Su, W.D., Yoshioka, H., Murakami, T., Arata, J., Ninomiya, Y.,2000. The brain link protein-1 (BRAL1): cDNA cloning, genomic structure, andcharacterization as a novel link protein expressed in adult brain. Biochem.Biophys. Res. Commun. 276, 982e989.

Ishihama, Y., Sato, T., Tabata, T., Miyamoto, N., Sagane, K., Nagasu, T., Oda, Y., 2005.Quantitative mouse brain proteomics using culture-derived isotope tags as in-ternal standards. Nat. Biotechnol. 23, 617e621.

Iwaki, T., Kume-Iwaki, A., Liem, R.K., Goldman, J.E., 1989. Alpha B-crystallin isexpressed in non-lenticular tissues and accumulates in Alexander’s diseasebrain. Cell 57, 71e78.

Iwaki, T., Wisniewski, T., Iwaki, A., Corbin, E., Tomokane, N., Tateishi, J., Goldman, J.E.,1992. Accumulation of alpha B-crystallin in central nervous system glia andneurons in pathologic conditions. Am. J. Pathol. 140, 345e356.

Jackson-Lewis, V., Przedborski, S., 2007. Protocol for the MPTP mouse model ofParkinson’s disease. Nat. Protoc. 2, 141e151.

Jenner, P., 1991. Oxidative stress as a cause of Parkinson’s disease. Acta Neurol.Scand. Suppl. 136, 6e15.

Jonsson, Z.O., Dhar, S.K., Narlikar, G.J., Auty, R., Wagle, N., Pellman, D., Pratt, R.E.,Kingston, R., Dutta, A., 2001. Rvb1p and Rvb2p are essential components of achromatin remodeling complex that regulates transcription of over 5% of yeastgenes. J. Biol. Chem. 276, 16279e16288.

Kloetzel, P.M., Soza, A., Stohwasser, R., 1999. The role of the proteasome system andthe proteasome activator PA28 complex in the cellular immune response. Biol.Chem. 380, 293e297.

Li, A., Guo, H., Luo, X., Sheng, J., Yang, S., Yin, Y., Zhou, J., Zhou, J., 2006. Apomor-phine-induced activation of dopamine receptors modulates FGF-2 expression inastrocytic cultures and promotes survival of dopaminergic neurons. FASEB J. 20,1263e1265.

Liu, G., Loraine, A.E., Shigeta, R., Cline, M., Cheng, J., Valmeekam, V., Sun, S., Kulp, D.,Siani-Rose, M.A., 2003. NetAffx: affymetrix probesets and annotations. NucleicAcids Res. 31, 82e86.

Mandel, S., Grunblatt, E., Riederer, P., Amariglio, N., Jacob-Hirsch, J., Rechavi, G.,Youdim, M.B., 2005. Gene expression profiling of sporadic Parkinson’s diseasesubstantia nigra pars compacta reveals impairment of ubiquitin-proteasomesubunits, SKP1A, aldehyde dehydrogenase, and chaperone HSC-70. Ann. N. Y.Acad. Sci. 1053, 356e375.

Masilamoni, J.G., Jesudason, E.P., Bharathi, S.N., Jayakumar, R., 2005a. The protectiveeffect of alpha-crystallin against acute inflammation in mice. Biochim. Biophys.Acta 1740, 411e420.

Masilamoni, J.G., Vignesh, S., Kirubagaran, R., Jesudason, E.P., Jayakumar, R., 2005b.The neuroprotective efficacy of alpha-crystallin against acute inflammation inmice. Brain Res. Bull. 67, 235e241.

McArthur, S., Cristante, E., Paterno, M., Christian, H., Roncaroli, F., Gillies, G.E.,Solito, E., 2010. Annexin A1: a central player in the anti-inflammatory andneuroprotective role of microglia. J. Immunol. 185, 6317e6328.

McGeer, P.L., Yasojima, K., McGeer, E.G., 2001. Inflammation in Parkinson’s disease.Adv. Neurol. 86, 83e89.

McNaught, K.S., Olanow, C.W., 2006. Protein aggregation in the pathogenesis offamilial and sporadic Parkinson’s disease. Neurobiol. Aging 27, 530e545.

Meffre, D., Delespierre, B., Gouezou, M., Leclerc, P., Vinson, G.P., Schumacher, M.,Stein, D.G., Guennoun, R., 2005. The membrane-associated progesterone-bind-ing protein 25-Dx is expressed in brain regions involved in water homeostasisand is up-regulated after traumatic brain injury. J. Neurochem. 93, 1314e1326.

Mizuno, Y., Yoshino, H., Ikebe, S., Hattori, N., Kobayashi, T., Shimoda-Matsubayashi, S., Matsumine, H., Kondo, T., 1998. Mitochondrial dysfunction inParkinson’s disease. Ann. Neurol. 44, S99eS109.

Oohashi, T.,Hirakawa, S., Bekku,Y., Rauch,U., Zimmermann,D.R., Su,W.D.,Ohtsuka, A.,Murakami, T., Ninomiya, Y., 2002. Bral1, a brain-specific link protein, colocalizingwith the versican V2 isoform at the nodes of Ranvier in developing and adultmouse central nervous systems. Mol. Cell Neurosci. 19, 43e57.

Renkawek, K., Stege, G.J., Bosman, G.J., 1999. Dementia, gliosis and expression of thesmall heat shock proteins hsp27 and alpha B-crystallin in Parkinson’s disease.Neuroreport 10, 2273e2276.

Shao, W., Zhang, S.Z., Tang, M., Zhang, X.H., Zhou, Z., Yin, Y.Q., Zhou, Q.B., Huang, Y.Y.,Liu, Y.J., Wawrousek, E., Chen, T., Li, S.B., Xu, M., Zhou, J.N., Hu, G., Zhou, J.W.,2013. Suppression of neuroinflammation by astrocytic dopamine D2 receptorsvia alphaB-crystallin. Nature 494, 90e94.

Shinohara, H., Inaguma, Y., Goto, S., Inagaki, T., Kato, K., 1993. Alpha B crystallin andHSP28 are enhanced in the cerebral cortex of patients with Alzheimer’s disease.J. Neurol. Sci. 119, 203e208.

Sijts, A., Sun, Y., Janek, K., Kral, S., Paschen, A., Schadendorf, D., Kloetzel, P.M., 2002.The role of the proteasome activator PA28 in MHC class I antigen processing.Mol. Immunol. 39, 165e169.

Simunovic, F., Yi, M., Wang, Y., Macey, L., Brown, L.T., Krichevsky, A.M.,Andersen, S.L., Stephens, R.M., Benes, F.M., Sonntag, K.C., 2009. Gene expressionprofiling of substantia nigra dopamine neurons: further insights into Parkin-son’s disease pathology. Brain 132, 1795e1809.

Venteicher, A.S., Meng, Z., Mason, P.J., Veenstra, T.D., Artandi, S.E., 2008. Identifi-cation of ATPases pontin and reptin as telomerase components essential forholoenzyme assembly. Cell 132, 945e957.

Wang, K., Zhang, J., Xu, Y., Ren, K., Xie, W.L., Yan, Y.E., Zhang, B.Y., Shi, Q., Liu, Y.,Dong, X.P., 2013. Abnormally upregulated alphaB-crystallin was highly

Y. Liu et al. / Neurobiology of Aging 36 (2015) 1686e1691 1691

coincidental with the astrogliosis in the brains of scrapie-infected hamsters andhuman patients with prion diseases. J. Mol. Neurosci. 51, 734e748.

Williams, D., Norman, G., Khoury, C., Metcalfe, N., Briard, J., Laporte, A., Sheibani, S.,Portt, L., Mandato, C.A., Greenwood, M.T., 2011. Evidence for a second messengerfunction of dUTP during Bax mediated apoptosis of yeast and mammalian cells.Biochim. Biophys. Acta 1813, 315e321.

Wood, M.A., McMahon, S.B., Cole, M.D., 2000. An ATPase/helicase complex is anessential cofactor for oncogenic transformation by c-Myc. Mol. Cell 5, 321e330.

Zheng, B., Liao, Z., Locascio, J.J., Lesniak, K.A., Roderick, S.S., Watt, M.L., Eklund, A.C.,Zhang-James, Y., Kim, P.D., Hauser, M.A., Grunblatt, E., Moran, L.B.,Mandel, S.A., Riederer, P., Miller, R.M., Federoff, H.J., Wullner, U.,Papapetropoulos, S., Youdim, M.B., Cantuti-Castelvetri, I., Young, A.B.,Vance, J.M., Davis, R.L., Hedreen, J.C., Adler, C.H., Beach, T.G., Graeber, M.B.,Middleton, F.A., Rochet, J.C., Scherzer, C.R., 2010. PGC-1alpha, a potentialtherapeutic target for early intervention in Parkinson’s disease. Sci. Transl.Med. 2, 52ra73.

1

Supplementary figures

Supplementary Fig. S1. Quantitative proteomic analysis of the substantia nigra of PD

patients and healthy controls. (A, B) The subcellular (A) and functional distributions (B) of the

229 proteins which were identified by mass spectrometry and presented in all the samples.

2

Supplementary Fig. S2. Upregulation of Cryab in reactive astrocytes and microglia in the SN

of MPTP mouse model of PD. (A) Immunohistochemical staining for Cryab in the ventral

mesencephalon of adult male mice administered with either saline or MPTP. Arrows indicate

the SN pars compacta (SNpc). Cryab is up-regulated in reactive glial cells in the SN of

MPTP-treated mice. Cryab-positive glial cells represent the classical glial scar in the SNpc

(arrows) of MPTP-treated mice. (B, C) Upregulation of Cryab in reactive astrocytes (B) and

microglia (C). Arrows indicate double-labeled cells. Scale bars, A, 100 m; B and C, 20 m.

3

Supplementary Table S1. Summary of the demographic and clinicopathological data on the

three Parkinson’s disease and three control cases used in this study

Number

of pairsAge Sex Post-mortem time Condition

1 69 Male 5 hrs and 55 min Healthy

68 Male 6 hrs PD

2 83 Female 5 hrs and 15 min Healthy

83 Female 5 hrs PD

3 71 Male 6 hrs Healthy

84 Male 5 hrs and 10 min PD

4

Supplementary Table S2. List of differential expressed proteins that showed more than

1.5-fold changes across all three matched pairs of samples.

Protein title Gene symbol Protein ID Average ratio

(PD / Control)*

Increased

Alpha crystallin B chain Cryab IPI00021369 2.49 ± 0.67

Hyaluronan and proteoglycan link protein 2 Hapln2 IPI00029184 8.69 ± 3.22

Coiled-coil domain-containing protein 94 Ccdc94 IPI00306471 3.11 ± 0.34

Membrane associated progesterone receptor

component 1 Pgrmc1 IPI00220739 2.55 ± 0.43

Heterogeneous nuclear ribonucleoprotein

U-like protein 2 Hnrnpul2 IPI00456887 5.28 ± 3.20

Proteasome activator complex subunit 2 Psme2 IPI00384051 1.68 ± 0.06

Decreased

DUT protein Dut IPI00375015 0.42 ± 0.04

Potassium channel tetramerization domain

containing protein 13 Kctd13 IPI00157833 0.22 ± 0.08

Tubulin alpha-1 chain Tba1 IPI00007750 0.49 ± 0.11

RuvB-like AAA ATPase 1 Ruvb1 IPI00021187 0.26 ± 0.01

Annexin A1 Anxa1 IPI00218918 0.25 ± 0.16

* denotes the average of fold-changes of a specific protein in the three pairs of PD and control

subjects which were used to generate the proteomic data (mean ± SEM, n=3).