Proteomics of Huntington's Disease-Affected Human Embryonic

Article pubs.acs.org/jpr

Proteomics of Huntington’s Disease-Affected Human Embryonic Stem Cells Reveals an Evolving Pathology Involving Mitochondrial Dysfunction and Metabolic Disturbances Leon R. McQuade,† Anushree Balachandran,‡,§ Heather A. Scott,‡ Simer Khaira,‡ Mark S. Baker,*,§,⊥ and Uli Schmidt‡,⊥ †

Australian Proteome Analysis Facility, §Australian School of Advanced Medicine, Macquarie University, Sydney, New South Wales 2109, Australia ‡ Genea Biocells, Genea, Lv. 2, 321 Kent Street, Sydney 2000, Australia S Supporting Information *

ABSTRACT: Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation in the Huntingtin gene, where excessive (≥36) CAG repeats encode for glutamine expansion in the huntingtin protein. Research using mouse models and human pathological material has indicated dysfunctions in a myriad of systems, including mitochondrial and ubiquitin/proteasome complexes, cytoskeletal transport, signaling, and transcriptional regulation. Here, we examined the earliest biochemical and pathways involved in HD pathology. We conducted a proteomics study combined with immunocytochemical analysis of undifferentiated HD-affected and unaffected human embryonic stem cells (hESC). Analysis of 1883 identifications derived from membrane and cytosolic enriched fractions revealed mitochondria as the primary dysfunctional organ in HD-affected pluripotent cells in the absence of significant differences in huntingtin protein. Furthermore, on the basis of analysis of 645 proteins found in neurodifferentiated hESC, we show a shift to transcriptional dysregulation and cytoskeletal abnormalities as the primary pathologies in HD-affected cells differentiating along neural lineages in vitro. We also show this is concomitant with an up-regulation in expression of huntingtin protein in HD-affected cells. This study demonstrates the utility of a model that recapitulates HD pathology and offers insights into disease initiation, etiology, progression, and potential therapeutic intervention. KEYWORDS: Huntington’s disease, human embryonic stem cells, neurodifferentiation, shotgun proteomics, immunocytochemistry, IPG-IEF, LC−MS/MS

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INTRODUCTION Huntington’s disease (HD) is a progressive neurodegenerative disease that manifests pathologically with the prominent loss of medium spiny neurons (MSN) of the striatum, culminating in organismal death. Symptoms appear in midlife and include chorea; cognitive decline; psychiatric disorders; and, most commonly, depression. HD is caused by a genetically dominant expansion of cytosine-adenine-guanine (CAG) residues in exon 1 of the Huntingtin gene, translating to a poly glutamine (polyQ) expansion in the N-terminus of the huntingtin protein (Htt). The length of the repeat correlates with the age of onset, with more than 40 CAG repeats being fully penetrant with adult onset while greater than 70 repeats generally results in juvenile onset.1 © XXXX American Chemical Society

The CAG repeat expansion responsible for HD was identified in 1993, and since then, researchers have concentrated on deducing the modus operandi for disease progression. 2 Hypotheses abound expounding both a toxic gain-of-function conferred to Htt through polyQ expansion or a loss of normal cellular function due to a reduction in wild-type Htt. Bioenergetic deficits, an inability to neutralize oxygen free radicals, alterations in brain-derived neurotrophic factor expression, toxicity of Nterminal Htt fragments due to caspase cleavage of the mutant protein, and altered mitochondrial calcium homeostasis are Received: June 27, 2014

A

dx.doi.org/10.1021/pr500649m | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

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Table 1. Properties and Analytical Details of Human Embryonic Stem Cells cell line f

GENEA002 GENEA019b GENEA022d GENEA023d GENEA029 GENEA017f GENEA018f GENEA020b GENEA046f GENEA089c,f GENEA090c,f GENEA091c,f

disease status

sex

pluripotency determined bya

unaffected unaffected unaffected unaffected Unaffected HD HD HD HD HD HD HD

M F M M F M F F F F F F

ICC, P, T ICC, P, T ICC, T ICC, T ICC, P ICC, P, T ICC, P, T ICC, P, T ICC, P, T ICC, P ICC, P ICC, P

Htt polyQ repeat

pluripotent ICC

neurodifferentiated ICC

15/21 18/21e 18/21e 22/22e 12/40 17/46 17/48 23/45 19/41 19/46e 21/42e

√ √ √ √ √ √ √ √ √ √ √ √

√ √ √ √ √ √ √ √ √ √ √ √

a

ICC, immunocytochemistry; P, PluriTest; T, teratoma. bGENEA019 and 020 are siblings. cGENEA089, 090, and 091 are siblings. dGENEA022 and 023 are siblings. eHtt polyQ repeat length determined by capillary electrophoresis. fApproved by the National Institutes of Health (NIH).

hypotheses currently under investigation.3 Despite this, the primordial source of HD pathology remains unknown,4 and treatment of patients currently is solely symptomatic and palliative. The first genetically modified murine model of HD was created in 1996, and a dozen or so have followed in the decades since.5 These models have been valuable in elucidating the pathophysiological mechanisms. Nonetheless, animal models have failed to completely mirror the pathology observed in humans or lead to discovery and validation of curative therapeutics.6 Monogenic disease-affected human pluripotent stem cells are becoming a disease model of choice, in part because of their unmodified genomes, relative cost-effectiveness, and unlimited capacity for self-renewal and differentiation into all somatic cell types. Several induced pluripotent stem cell (iPSC) lines and hESC lines now exist which naturally carry, on at least one allele, >40 Htt polyQ repeats in exon 1 of the Huntingtin gene (mutant Htt).7,8 Because the clinical manifestations of HD often do not present until midlife, the ability of postmitotic somatic cells created from stem cells to mimic HD pathology seemed questionable; however, several groups have now shown that neural cell types derived from HD-affected pluripotent stem cells undergo changes reflective of HD pathology, making them useful tools for both basic research and drug discovery.9−14 Few transcriptomic or proteomic studies have investigated if HD-affected pluripotent human embryonic stem cells (hESC) or iPSC recapitulate any aspects of HD pathology.10,12,15,16 Here, we detail a proteomics study comparing unaffected and HDaffected hESC as well as terminally differentiated neural cells derived from both hESC cell types. This experimental design allows for an objective examination of HD progression using an in vitro human system. Proteomics has advanced to the point that it is now recognized that the scientific question being asked will need specific workflows and that the nature of the proteome being investigated (for example, cytosolic or membrane proteomes) may well require the application of customized techniques.17 Because this study was one of the first to examine the proteome of HD affected human embryonic stem cells, the design aimed at getting maximum coverage of both membrane-bound and cytosolic proteins using label-free proteomics, now recognized as a highthroughput method for quantitative clinical proteomics.18 The choice of label-free or labeling-based approaches (SILAC, iTRAQ, or TMT) have recently been examined and reviewed,18−20 and it was found that the highest proteome

coverage and the largest dynamic range is achieved using labelfree approaches. The procedure for this study involved a “shotgun” approach in which isolated proteins are trypsin-digested in the presence of methanol, followed by peptide filtering using immobilized pH gradient isoelectric focusing (peptide IPG-IEF) in front of reversed phase liquid chromatography tandem mass spectrometry (LC − MS/MS). Such preparation and filtration has been reported to minimize false positive identifications while increasing protein identification coverage including difficult to analyze cellular components such as membranes.21−25 This approach was conducted on triplicate cell lines over all conditions to increase statistical robustness. We used quantitation by normalized spectral abundance factors, introduced in 2006,26 as it is straightforward to implement, widely used and referenced (350+ citations), and additionally we have an established analysis pipeline for this type of quantitation in the Australian Proteome Analysis Facility.27 Our results based on interpreting expression status of high quality reproducible protein identifications demonstrate that mitochondrial dysfunction is the most profound of multiple early manifestations found in pluripotent HD-affected hESC. This biochemical disruption induces or cascades to transcriptional dysregulation with cytoskeletal disturbances as cells differentiate along neural lineages.

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EXPERIMENTAL SECTION

Cell Lines

Embryo donation, stem cell line derivation, and culture was performed as previously described,7 and all relevant research protocols were in compliance with international guidelines including the U.S. National Acadamies’ 2008 Guidelines for Human Embryonic Stem Cell Research. Research conducted was approved by the Genea Human Ethics Committee, an independent committee operating and constituted according to the requirements of Australia’s National Health and Medical Research Council (NH&MRC). We performed proteomic analyses of undifferentiated hESC on cell lines GENEA019 (unaffected; Huntingtin polyglutamine exon 1 allele repeat lengths, 15/21) and GENEA046 (HD-affected; 23/45). Proteomic analysis on neurodifferentiated hESC was conducted by pooling neurodifferentiated cells from unaffected cells lines GENEA019 (15/21), GENEA 23 (18/21), and GENEA029 (22/22) and HD-affected cell lines GENEA018 (17/46), B



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Figure 1. Proteomics workflow. Standardized workflow for the retrieval of proteins from cells and isoelectric peptide focusing.

Cell Culture

GENEA020 (17/48), and GENEA046 (23/45). This provided sufficient protein for proteomic analysis following neurodifferentiation and additionally reduces potential genetic bias. For immunocytochemical (ICC) analysis, additional cell lines were used (GENEA002, GENEA017, GENEA022, GENEA023, GENEA029, GENEA089, GENEA090, and GENEA091). Cell line details are listed in Table 1.

For proteomic analysis of hESC, GENEA019 and GENEA046 were expanded separately in triplicate as previously described.30 Differentiation along the neuroderm lineage was induced as reported previously.28 Briefly, cells were exposed to Neurobasal Media (Life Technologies) supplemented with 100 ng/mL each of the proteins Noggin and Brain Derived Neurotrophic Growth Factor (R&D Systems) and additional components currently commercial-in-confidence to Genea Biocells. Cells were differentiated and maintained in culture until they reached a mature morphology, typically after 40−45 days and judged by the expression of more mature markers, including neurofilament 250 and loss of cells expressing progenitor markers, including nestin (data not shown). Cells were then harvested for ICC analysis and frozen as a pellet at −80 °C for proteomics. Culture conditions and media employed were identical for the culturing of both pluripotent and neurodifferentiating cells. All cell lines and differentiated neurons are commercially available from Genea Biocells.

Molecular Characterization

The number of CAG repeats (as well as adjacent non-HD related poly proline CCG repeats) in exon 1 of the Huntingtin gene was determined by PCR amplification (5′-CCTTCGAGTCCCTCAAGTCCTTC-3′ and 5′-TGAGGCAGCAGCGGCTGT-3′) followed by capillary electrophoresis using Life Technologies 3130 Genetic Analyzer. CAG repeat number was calculated by comparison to known synthetically synthesized standards. This method assumes the CCG repeat size is seven.29 CAG-CCG amplicons from GENEA019, GENEA017, GENEA018, GENEA020, GENEA046, and GENEA089 were sequenced by the Australian Genome Research Facility, Melbourne, Australia. C



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Figure 2. HD-affected and unaffected hESC pluripotency and response to cellular toxicity. ICC image of (a) HD-affected hESC and (b) unaffected hESC stained with markers of pluripotency. ICC images of hESC stained with Oct4 and Htt (c) HD-affected and (d) unaffected. (e) Box plot representing median and interquartile ranges showing a significant difference in pluripotency marker Lin-28 (unaffected, n = 112 cultures; HD, n = 135 cultures) between HD-affected and unaffected hESC, but no difference in Nanog (unaffected, n = 112 cultures; HD, n = 135 cultures) or Oct4 (unaffected, n = 112 cultures; HD, n = 135 cultures). (f) Box plot representing median and interquartile ranges showing no difference in Htt expression between HD-affected (n = 70 cultures) and unaffected (n = 70 cultures) hESC. (g) Box plot representing median and interquartile ranges showing a significant difference between the STS treated (unaffected, n = 112 cultures; HD, n = 135 cultures) and untreated (unaffected, n = 106 cultures; HD, n = 128 cultures) for both HD-affected and unaffected hESC. In all cases, “cultures” refers to wells in a microtiter plate.

applied for 1 h at 25 °C. Cells were imaged using an IN Cell Analyzer 6000 (GE-Healthcare) and analyzed using standard high-content analysis methodology (Developer Toolbox v1.9 software package) whereby hESC were identified by drawing a circular mask determined by Hoechst staining and then identifying positive cells when Nanog, Oct4, and Lin-28 staining overlapped with an identified nucleus. For neurons, MAP2 expression was utilized to draw cellular masks that could be analyzed for the properties of fiber length, number of end nodes, and number of branch nodes. Statistical analyses were performed on the average of these measurements. To determine significance, the Mann−Whitney U test was employed for the analysis of cell populations because this does not assume a normal data distribution. Alpha was set at 0.05. Analysis of Htt protein expression levels was performed by measuring average pixel intensity within individual cells identified by high-content image segmentation (Developer Toolbox v1.9). This type of measurement usually follows a normal distribution and a t-test was applied for statistical analysis

Immunocytochemical (ICC) Analysis

hESC and neurodifferentiated cells were plated on collagen and polyornithine/laminin-coated 96-well plates (PerkinElmer), respectively. Cells were washed with phosphate-buffered saline (Life Technologies) and fixed with 10% formalin (Sigma) for 10 min. hESC were stained for pluripotency markers PE-conjugated mouse anti-Nanog (BD Pharmingen 560483, 1:150), Alexa-647conjugated mouse anti-Oct4 (BD Pharmingen 560307, 1:200), and DyLight488-conjugated rabbit anti-Lin-28 (Abcam ab139910, 1:150). Differentiated cells were stained with the neurodifferentiation markers chicken antihuman MAP2 (Covance PCK-554P, 1:17 000), mouse antihuman GAD65 (Abcam ab26113, 1:125), and rabbit antihuman GABA (Sigma A2052, 1:275) to quantify the extent of neurodifferentiation. Pluripotent and neurodifferentiated cells were also stained with rabbit antihuman huntingtin (antibodies online ABIN350376, 1:500). Nuclei were visualized with Hoechst 33342 (Invitrogen H3570, 1:5000). Alexaconjugated secondary antibodies (Invitrogen, 1:500) were D



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Figure 3. HD-affected and unaffected neural cell properties and response to cellular toxicity. ICC image of cultures stained with markers of mature neurons (a) HD-affected neural cells and (b) unaffected neural cells. ICC images of cultures stained for Htt (c) HD-affected neural cells and (d) unaffected neural cells. (e) Box plot representing median and interquartile ranges showing a significant difference in GABAergic neuron marker GAD65/67 (unaffected, n = 91 cultures; HD, n = 136 cultures) between HD-affected and unaffected neurons, but no difference in neuronal marker MAP2 (unaffected, n = 91 cultures; HD, n = 136 cultures). (f) Box plot representing median and interquartile ranges showing the proportion of GAD65/ 67+ cells for each cell line. (g) Properties of neurons (unaffected, n = 90 cultures; HD, n = 135 cultures) showing a significant up-regulation in the number of branch nodes and end nodes in HD-affected cultures with no change in fiber length. (h) Box plot representing median and interquartile ranges showing a significant difference in Htt expression between HD-affected (n = 84 cultures) and unaffected (n = 48 cultures) neural cells. (i) Box plot representing median and interquartile ranges showing a significant difference between the STS treated (unaffected, n = 98 cultures; HD, n = 148 cultures) and untreated (unaffected, n = 102 cultures). In all cases “cultures” refers to wells in a microtiter plate.

(Figure 2f, Figure 3h). All statistical analyses were conducted using IBM SPSS v21. Staurosporine (STS) is a broad spectrum inhibitor of protein kinase activity and is generally employed to induce apoptosis in vitro.31 HD-affected and unaffected hESC were treated with 20 nM STS for 6 h to determine if cells were differentially

susceptible to STS-induced apoptosis, measured as loss of cell density. Sample Preparation for Proteomics

The specialized proteomics workflow has been reported previously30 (Figure 1). Harvested cells frozen at −80 °C were thawed and lysed either via sonication or using commercially E



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available protein extraction reagents (Pierce). Modern in-depth proteomic analysis is still limited by the amount of available starting materials and by losses incurred during each extraction step. In cellular based analyses, the yield of protein is dependent on cell numbers; an average 1 × 107 cells per line hESC and 5 × 106 cells per neurodifferentiated cell line was used in this study. Therefore, we employed different strategies for protein extraction for the different cell types. Sufficient protein was retrieved from hESC to separate into predominantly membrane and cytoplasmic fractions, which enabled peptide focusing over pI ranges, 3−10 (broad range) and 4−7 (narrow range). We retrieved membrane protein enriched fractions as previously described30 while nonprecipitated proteins in the sodium bicarbonate (pH 11) supernatant were retrieved by reducing starting volumes to ∼1.0 mL via vacuum centrifugation, followed by 24 h buffer exchange into ammonium bicarbonate (pH 7.8) using dialysis cassettes (2.0 kDa MWCO, 0.5−3 mL capacity; Slide-A-Lyser; Pierce), followed by reduction and alkylation as previously described.23,30 Because of low cell numbers from neurodifferentiated cell lines, whole cell lysates were pooled into HD-affected and unaffected lots with equal protein amounts. Peptides were focused using only the pI 3−10 range strips. To enhance statistical robustness, triplicate analyses were conducted on extracted proteins from all cell lines with culturing, differentiation, harvesting, and protein extraction being conducted in parallel. Peptides were recovered after focusing by cutting each strip into 24 equally sized pieces, eluting, and desalting. Peptides from each of the 24 fractions were analyzed by reversed phase nano LC−MS/MS as routinely deployed in APAF and as previously described.23,30

submitted to Ingenuity Pathway Analysis (IPA; www.ingenuity. com) to determine if the manual assignation of differentially expressed proteins to biological processes was still relevant against a search of broader scientific literature and to assess if those cellular processes are significantly perturbed in HDaffected cells. All data analysis was carried out within the R statistical programming environment.

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RESULTS

Unaffected and HD-Affected hESC are Pluripotent, Express Htt, and Demonstrate an Equitable Response to Cellular Stress

Teratoma studies, ICC, and Pluritest32 showed stem cells from the five unaffected and six HD-affected hESC lines utilized in this study to be pluripotent (Table 1). In agreement with other studies, no significant difference was found between HD-affected and unaffected hESC for expression of Nanog (Mann−Whitney U z = −1.411, p = 0.158) or Oct4 (Mann−Whitney U z = −0.467, p = 0.641)14 (Figure 2a−d). Cytoplasmic pluripotency marker Lin-28 was significantly upregulated in HD compared with unaffected hESC (Mann−Whitney U z = −6.77, p < 0.001; Figure 2e); however, individual cell lines do vary in growth rate and cell density. Because Lin-28 expression was found to be significantly negatively correlated with cell density (n = 247, r = −0.402) and HD-affected hESC were found to be less dense than unaffected hESC (Mann−Whitney U z = −4.10, p < 0.001), the increase in Lin-28 positive cells in HD is considered to result from lower cell density. Comparing the average pixel intensity of the cytoplasmic area for Htt protein expression found no significant difference between HD-affected and unaffected hESC (Figure 2f). HD-affected and unaffected hESC treated with 20 nM STS for 6 h showed no difference; however, both groups had significantly reduced cell density compared with their untreated equivalents (Mann−Whitney U z = −3.69, p < 0.001; Mann−Whitney U z = −2.21, p = 0.027, respectively; Figures 2g), although there is no significant difference in the sensitivity to cellular stress between HD-affected and unaffected cells.

Proteomic Data Analysis

Protein identifications were made using The Global Proteome Machine (GPM; www.thegpm.org). The X!Tandem protein identifications files with log(e) values less than −1 were merged for all gel fractions and from the corresponding membrane and cytoplasmic fractions of the hESC analyses. This resulted in one combined protein identification file for each replicate. Protein identifications were filtered to retain only reproducibly present proteins, namely, proteins found in all replicates of at least one cell line, and further having an average spectral count of at least two counts for at least one sample. The resulting raw spectral counts were converted to normalized spectral abundance factors (NSAF).26 Specifically, the spectral counts were divided first by the estimated protein length to account for expected differences in number of identified peptides from longer proteins. Then normalized counts were divided by the total sum of the spectral counts/length to account for differences in total sample amount. The distribution of log(NSAF) data was examined using boxplots and kernel density plots (data not shown) and was deemed suitable for subsequent statistical analyses, although the total protein counts were lower for the HD-affected lines. The full list of identified proteins is presented in Supporting Information (SI) Table S1. Average fold changes between cell lines were based on the NSAF values, and proteins differentially expressed between the two cell lines were identified by a t-test comparison of the triplicate log NSAF values, retaining proteins with a p-value less than 0.05. The functions of these proteins were determined by a manual search of available scientific literature and protein databases. Biological processes that involve two or more of these proteins were noted in expectation that these processes might be disrupted in HD-affected cells. These proteins were also

HD-Affected hESC Demonstrate down-Regulation of Key Proteins Involved in the Electron Transport Chain (ETC) and Oxidative Stress

Quantitative proteomic analysis of triplicate pluripotent HDaffected and unaffected hESC lines derived from membrane and cytosolic enriched fractions yielded 1883 high quality reproducible protein identifications. Of these, 61 proteins were upregulated and 225 were down-regulated in HD-affected hESC. Pathway analysis using IPA identified mitochondrial dysfunction as the top canonical pathway (p = 2.13 × 10−9), with 15 proteins involved in that pathway significantly altered (SI Table S2). HD-affected hESC demonstrates reductions in expression levels of components of ETC complexes I, III, and IV as well as loss of NADH and fatty acid oxidation capacity. A functional ETC is required for ATP generation, intracellular Ca2+ buffering, regulation of apoptosis, and minimization of reactive oxygen species (ROS) generation. The noted changes in expression of proteins from complex I (NADH dehydrogenase 1 alpha subcomplex 4, NADH dehydrogenase Fe−S protein 8), complex III (ubiquinol−cytochrome c reductase Rieske Fe−S polypeptide 1, ubiquinol−cytochrome c reductase core protein I and II), and complex IV (cytochrome c oxidase polypeptide VIIa polypeptide 2, cytochrome c oxidase II) collectively imply a major lack of proton motive force in the mitochondria of HD-affected hESC. F



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Table 2. Direct Components of ETC Complexes Differentially Expressed in HD-Affected hESC Identified by Ingenuity IPA Analysis function

p-value

fold change (HD/ unaffected)

transfers electrons from reduced cytochrome c to oxygen, terminal enzyme of the ETC, complex IV transfers electrons from NADH to ubiquinones, essential component of complex I of the ETC binds two iron−sulfur clusters of complex I, required for electron transfer transfers eletrons from cytochrome c to catalytic subunit 1, terminal enzyme of the ETC, complex IV, mitochondrially encoded key subunit of complex III

0.0120

−5.65

0.0139

−5.61

0.0264

−4.41

0.0406

−3.10

0.0234

−3.09

0.0210

−2.83

0.0393

−2.60

0.0021

−2.00

0.0308

−1.59

Ensembl ID ENSP00000

protein description cytochrome c oxidase polypeptide VIIa polypeptide 2 (COX7A2)

359106

NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 (NDUFA4)

339720

NADH dehydrogenase (ubiquinone) Fe−S protein 8 (NDUFS8)

315774

mitochondrial encoded cytochrome c oxidase II (MT-CO2)

354876

ubiquinol−cytochrome c reductase, Rieske iron−sulfurpolypeptide 1 (UQCRFS1) ubiquinol−cytochrome c reductase core protein I (UQCRC1)

306397

ubiquinol−cytochrome c reductase core protein II (UQCRC2)

268379

ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1 (ATP5AT) ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B)

282050

203407

262030

nuclear encoded component of complex III, transfers electrons from coenzyme QH2 to ferricytochrome c nuclear encoded component of complex III, transfers electrons from coenzyme QH2 to ferricytochrome c F1 complex is responsible for catalytic binding of ADP and Pi to create ATP F1 component consists of 3 alphas, 3 betas, and a single of either gamma, delta, or epsilon

Table 3. Indirect Components of Mitochondrial Function Differentially Expressed in HD-Affected hESC Identified by Ingenuity IPA Analysis protein description cytochrome b5 reductase 3 (CYB5R3) apoptosis-inducing factor mitochondrial (AIF) carnitine palmitoyltransferase 1A (CPT1) glycerol-3-phosphate dehydrogenase 2 (mGPDH) hydroxysteroid (17β) dehydrogenase 10 (HSD10) glutathione reductase (GSR)

Ensembl ID ENSP00000 379597 287295 365803 308610 168216 221130

p-value


responsible for NADH-dependent Q10 reduction, provides protection against extracellular oxidants apoptogenic and oxidoreductase functions, may interact with complex III

0.0050

−11.80

0.0379

−5.80

catalyzes the formation of acylcarnitine from carnitine and acyl-CoA, ratelimiting step of fatty acid oxidation FAD-dependent converter of glycerol-3-phosphate to dihydroxyacetone phosphate, electron donor for Q10 involved in isoleucine metabolism, required for the maintenance of mitochondrial structure reduces glutathione disulfide and NADPH to glutathione and NADP+, important mitigator of oxidative stress

0.0482

−5.01

0.0407

−3.35

0.0024

−2.69

0.0296

3.68

function

These changes should concomitantly result in an inability to synthesize ATP. In addition, direct reduction of the α and β subunits of the F1 complex of ATP synthase (ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1 [ATP5A1] and ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide [ATP5B]; Table 2) also signifies a decrease in the amount of ATP available in HD-affected cells. In addition to these ETC complex I components, three other reductases (cytochrome b5 reductase [CYB5R3], glycerol-3phosphate dehydrogenase 2 [mGPDH] and apoptosis inducing factor [AIF]) were found to be significantly decreased in HDaffected hESC (Table 3). Three proteins involved indirectly in mitochondrial function were also found to be significantly altered in HD-affected hESC (Table 3). In detail, both carnitine palmitoyltransferase 1 (CPT1) and hydroxysteroid 17β dehydrogenase 10 (HSD10) were downregulated, whereas glutathione reductase (GSR) was upregulated. Collectively, these data (Tables 2, 3, SI Table S3) demonstrate a significant disturbance in mitochondrial function and energy metabolism in the earliest pluripotent stage of HDaffected cells. Recently published data on the role of mitochondrial dysfunction in HD, Alzheimer’s, Parkinson’s, and amyotrophic

lateral sclerosis propose dynamin-related protein 1 (Drp1) and mitochondrial fission and fusion proteins (Fis1, Mfn1, Mfn2, and Opa1) as essential for mitochondrial fission/fusion balance and in provision of necessary ATP to neurons.33 Three of these (Drp1 (DMN1L), Fis1 (FIS1), and Mfn1 (MFN1)) were identified in both pluripotent and neurodifferentiated hESC with downregulated expression in HD-affected cells. In addition, Drp1 showed increased downregulation in the transition from the pluripotent to neurodifferentiated state. (SI Table S4). The significant role that this protein plays in the HD phenotype was recently illustrated when a peptide inhibitor (P110-TAT) was shown to reduce mitochondrial fragmentation and improve mitochondrial function and cell viability in neural cells derived from HD-affected human iPSC.34 In addition to its role in mitochondrial fragmentation, Drp1 is associated with other cellular functions, such as phosphorylation, peroxisomal fragmentation, ubiquitination, and cell death.33 Perturbations to the protein ubiquitination pathway in HD have been reported in both mouse and human studies. One of the earliest noted disruptions in the protein ubiquitination pathway came from a proteomics study of development in R2/6 HD mice.35 In the current study, IPA analysis of both pluripotent and neurodifferentiated HD-affected hESC proteins identified the G



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H1 family of proteins were significantly upregulated in HD neural cultures (Table 4).

ubiquitination pathway as being significantly disturbed (pluripotent: p = 0.00129 with 11 proteins affected, rank = 9; neurodifferentiated: p = 0.01202, with seven proteins affected, rank = 8). These analyses further strengthen the concept of the compounding effect of mitochondrial dysfunction on a variety of cellular processes in HD.

Table 4. Up-Regulation of Histone H1 Proteins in HD Compared with Unaffected Neural Cells

HD-Affected Neural Cells Show Increase in Neuronal Branch Nodes, an Up-Regulation in Htt Expression, and a Sensitivity to Cellular Toxicity

protein description histone cluster 1, H1a (HIST1H1A) histone cluster 1, H1b (HIST1H1B) histone cluster 1, H1c (HIST1H1C) histone cluster 1, H 1d (HIST1H1D) histone cluster 1, H1e (HITS1H1E) histone cluster 1, H1t (HIST1H1T)

hESC from four unaffected and six HD-affected lines were neurodifferentiated (approximately 40−45 days) before being analyzed by ICC to determine the number of GABAergic neurons present (Figure 3a−d). HD-affected and unaffected neural cultures have comparable proportions of MAP2+ cells; however, HD-affected cultures have a significantly higher proportion of GABAergic neurons (Mann−Whitney U z = −3.248, p = 0.001; Figure 3e). Examination of this expression in each cell line (Figure 3f) showed a substantial amount of variability in GAD65/67 expression. It is unknown if this phenomenon is a disease phenotype, a consequence caused by the inherent properties of any given cell line or limitations of the differentiation process. Further analysis of the morphological properties of neurons (MAP2+ cells) found no difference in the length of the neuronal fibers with disease state (Figure 3g). HDaffected neurons did have a significant increase in the average number of branch nodes (Mann−Whitney U z = −2.544, p = 0.011) and end nodes (Mann−Whitney U z = −2.573, p = 0.010) compared with unaffected neurons. A significant increase in branch and end nodes without a concurrent increase in fiber length indicated that HD-affected neurons were highly branched but less elongated than their unaffected counterparts. Htt protein expression, using the average pixel intensity of cytoplasmic areas, showed a significant increase in expression in HD-affected neural cells (t = −4.16, df =122, p < 0.001; Figure 3h). Although the accumulation of mutant Htt (polyQ expansion) has been reported as a developmental consequence of HD, the composition of wild type versus mutant Htt could not be determined because of the lack of specificity of the antibody employed in this study. None of the available antimutant huntingtin antibodies showed sufficient selectivity for mutant over wild type huntingtin in our cells. The reason may be that mutant-specific antibodies are raised against peptides containing >60 glutamine residues. Other studies commonly use more artificial models with very long CAG repeats, whereas our cell lines have CAG repeat lengths that are more representative of the “average” HD patient and therefore more clinically relevant. Treatment of neural cells with the nonspecific protein kinase inhibitor STS at 20 nM resulted in only HD-affected cultures suffering significant cell death, as determined by reduction in cell density (Mann−Whitney U z = −2.72, p = 0.007; Figure 3i). This demonstrates a clear susceptibility of HD-affected neural cultures to cellular stress that is not equivalent to that observed with unaffected counterparts.

Ensembl ID ENSP00000

p-value


244573

0.0190

2.36

330074

0.0166

1.86

339566

0.0015

1.94

244534

0.0159

2.04

307705

0.0020

2.46

341214

0.0003

52.74

Seven actin cytoskeletal signaling proteins were significantly altered (p = 5.22 × 10−4; Table 5). Neural cells generated from HD-affected hESC demonstrated a vast network of cytoskeletal protein alteration and dysregulation, presumably leading to an overall reduction in cell motility and intracellular trafficking. Decreases in the cytoskeletal proteins radixin (RDX), myosin light chain 6 (MYL6) and IQ motif-containing GTPase activating protein 1 (IQGAP1) all suggested that HD neuronal cells were undergoing severe disruptions in axonal transport.36−39 Talin-1,40 vinculin,41 and α-actinin42 are known to be intimately involved in the creation and maintenance of focal adhesions, the anchoring of cytoskeleton to the extracellular matrix (ECM) and critical for cell motility/migration, as well as response to the ECM environment. Reductions of profilin143 is associated with impaired formation of focal adhesions. Concomitant reductions of talin and α-actin would result in intensifying the ablation of cell-ECM interactions in HD-affected neural cells. The significant changes to expression levels in this collection of proteins, corroborated by ICC data on increased numbers of branch and end nodes, implied profound effects for neuronal anatomy and physiology in early HD neurological development.

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DISCUSSION The results obtained in this comprehensive study provide substantial evidence that HD hESC undergoes complex pathological changes highly consistent with reported clinical observations from HD patients and mouse models. It also provides a list of protein pathway targets that will contribute to current ongoing investigation into HD drug discovery.44 One of the advantages proteomics provides is the ability to globally examine biochemical pathways and the effects a single gene perturbation has on numerous cellular systems. A limitation of stem cell-based disease models in general is genetic variability between cell lines that may bias the results. The most pertinent way to address this is the use of genetically engineered isogenic controls; however, gene targeting efforts using sequence-directed nucleases have been unsuccessful because of the repetitive sequence and instability of the CAG repeat expansion causing Huntington disease (Dr. J. Arjomand, personal communication). To address this, hESC lines for proteomics studies were carefully matched on the basis of their cell biological properties (proliferation rate, gender, marker expression profiles, and differentiation propensities), and samples of all neurodiffer-

Cytoskeletal Abnormalities and Chromatin Modification in HD-Affected Neural Cells

Analysis of proteins isolated from four HD-affected and four unaffected neural cell cultures yielded 645 proteins with high quality reproducible identification. Of these, 136 proteins were significantly differentially expressed in HD-affected neural cells. IPA found the histone H1 family (granzyme A signaling; p = 3.3 × 10−9) as the top canonical pathway. Six members of the histone H



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Table 5. Dysregulation of Cytoskeletal Signalling Proteins in HD Compared with Unaffected Neural Cells

protein description radixin (RDX) myosin, light chain 6 (MYL6) actinin, alpha 4 (ACTN4) profilin 1 (PFN1) IQ motif containing GTPase activating protein 1 (IQGAP1) talin-1 (TLN1) vinculin (VCL)

function

p-value

fold change (HD/ Normal)

plays a crucial role in the binding of the barbed end of actin filaments to the plasma membrane acts as a linker between the head and tail of myosin, the head binds to actin filaments and is the site of ATP hydrolysis, the tail domain interacts with cargo molecules involved in binding actin to the membrane, thought to be involved in metastatic processes binds to actin and affects the structure of the cytoskeleton associates with calmodulin and serves as an assembly scaffold for the organization of a multimolecular complex that interfaces incoming signals to reorganize the actin cytoskeleton at the plasma membrane; promotes neurite outgrowth involved in connections of major cytoskeletal structures to the plasma membrane cytoskeletal protein associated with cell−cell and cell−matrix junctions, thought to function as one of several interacting proteins involved in anchoring F-actin to the membrane

0.0037 0.0210

−19.13 −8.40

0.0401 0.0438 0.0219

−7.48 −3.51 −3.15

0.0046 0.0002

−2.36 34.23

Ensembl ID ENSP00000 384136 301540 252699 225655 268182 316029 415489, 211998

biochemical targets (e.g., lipids, proteins, enzymes) and is a significant cause of cell death. Mitochondria are responsible for both the production (via inefficient electron flow through complex I and II) and clearance of ROS by Mn superoxide dismutase, coenzyme Q10 (ubiquinone), and glutathione.46 Like the components of the ETC listed in Table 2, the loss of these reductases in HD-affected hESC would likely have significant detrimental effects on the generation of the proton motive force and concomitantly place cells under considerable oxidative stress. Three proteins involved indirectly in mitochondrial function were also found to be significantly altered in HD-affected hESC compared with unaffected cells (Table 3), namely, CPT1, HSD10, and GSR. CPT1 transports fatty acids across the outer mitochondrial membrane as the rate-limiting step of fatty acid oxidation, the process by which free fatty acids are broken down into acetyl-coA to produce FADH2 and NADH (key electron donors of the ETC) via the citric acid cycle when glycogen is low.47 A reduction in CPT1 in HD-affected cells could reduce the number of long chain fatty acids available for energy production, thereby decreasing starting material for the ETC, culminating in reduced synthesis of ATP. Several other key components of the citric acid cycle were also significantly reduced in HD hESC (SI Table S3). Although HD-affected and unaffected hESC demonstrate similar properties with respect to pluripotency marker expression, Htt expression, and response to an inducer of apoptosis (STS), we found early signs of metabolic perturbation. These included impairment to both ETC and ROS detoxification in HD-affected hESC. We show that HD hESC in their unprimed, undifferentiated pluripotent state have severe reductions in antioxidant producing reductases and components of the citric acid cycle that parallel those previously reported in the HD R6/2 mouse model.48 What the current study demonstrates is that HD-affected hESC will be particularly susceptible to increasing energy demands during differentiation. In our study, neural cells varied with disease state, neuronal morphology, Htt expression, reaction to cellular stress and in their ability to produce GABAergic neurons. In addition, upregulation of six of the 11 members of the histone H1 family of proteins in HD-affected neural cultures (Table 4) insinuates transcriptional repression in HD-pathology as a potential target for development of next-generation disease-mitigating therapeutics. Currently, 138 histone deacetylase (HDAC) inhibitors are in phase I/II clinical trial, and six are in phase III trial across the U.S., Canada, and Australia (http://www.clinicaltrials.gov). Although the exact effect on gene expression produced by an

entiated cells were pooled in two groups of unaffected and HDaffected populations. This study found 286 differentially regulated proteins between HD-affected and unaffected hESC and 136 differentially regulated proteins in the neurodifferentiated derivatives produced from the same HD or control precursors. These data represent one of the first “global” proteomic investigations of HD in hESC. In Chae et al.10 only 26 proteins with ≥2 fold change were found to be differentially expressed in a comparison of pluripotent hESC and iPSC based solely upon 2-DE gel methodology. Their finding that an upstream p53-mediated apoptosis inducer, basic transcription factor 3 (BTF3), was upregulated in HD-affected iPSC was not reiterated in the current study. BTF3 was found in both undifferentiated and neurodifferentiated, HD-affected and unaffected hESC with nonsignificant p values for expression level fold changes (undifferentiated = 0.507; neurodifferentiated = 0.378) in the current study. This highlights either a difference between hESC and iPSC or the possible need to undertake replicate proteomic analyses as was undertaken here. Induction of apoptosis by the broad-based protein kinase activity inhibitor (STS) here did impact either HD-affected and unaffected hESCs compared with nontreated controls, as assessed by cell density. Importantly, however, neurodifferentiated HD-affected hESC were significantly more affected than non-HD-affected cells. Chae et al. concluded that processes, including programmed cell death, oxidative stress, and cellular oxygen-associated proteins, would be among the biological processes affected in HD iPSC. Although 23/26 proteins and all biological processes identified by the Chae et al. study were reiterated in the current study, our findings suggest that disrupted bioenergetics arising from dysfunctional mitochondria are the precursors for other cellular perturbations. This implies that the HD mutation (polyQ expression) impinges on mitochondrial structure and function during embryogenesis. Mitochondrial deficits have long been implicated in the pathophysiology of HD. Prior to its genetic cause being identified, observations that inhibition of complex II of the electron transport chain by 3-nitropropionic acid resulted in selective degeneration of the striatum, specifically GABAergic MSNs, led to the adoption of this toxicity as a popular model for HD.45 The reduction of key enzymes in HD-affected hESC compared with unaffected cells strongly suggests that energy metabolism in a HD patient is impaired from conception, prior to onset of any overt clinical symptom. Imbalance between reactive oxygen species (ROS) generation and antioxidants leads to extensive oxidative damage of key I



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significantly enriched in the Ratoviski study and showed a fold change of 5.799 (p = 3.792 × 10−02) in pluripotent hESC here. Three (ATP5A1, ATP5B, and ATP5F1) are subunits of ATP synthases, and RPS7 is involved with RNA-binding regulators of RNA processing and transcription. Such corroborative findings should start to resolve the questions of “cause and effect” in HD.55 High-quality, quantitative, proteome-wide data sets from HD and control, pluripotent, and neuronally differentiated cells cultured using the same media and culture conditions provide an unprejudiced insight into the pathology of Huntington’s disease. The data presented is strengthened by the additional effort applied to mine the full cellular proteome, especially the technically difficult proteome of the cell membrane. Using Gene Ontology annotation from the Ensembl database, categorizing for “membrane” and “plasma membrane” and using the reported R package56 revealed that in undifferentiated hESC, 35.8% of identified proteins were classified as membrane proteins, of which 16.8% were classified as plasma membrane proteins. For the neurodifferentiated hESC, 27.7% were identified as membrane and 16.2% as plasma membrane proteins. These numbers agree well with the estimates of Ahram et al.57 The data from this study suggests that depending on differentiation status, different biological pathways can be altered with mitochondrial dysfunction as the only or one of the earliest embryonic initiators precipitated via protein−protein interactions involving mutant (polyQ expanded) Htt. The identification of new, and confirmation of known biochemical pathways should help prioritize ongoing research into novel therapeutic strategies with alleviation of symptomology and disease progression as their focus.

increase in H1 expression is unknown, the overall outcome is likely to be a decrease in transcription of a myriad of genes.49 As a result of the interest in HDAC inhibitors for treatment of neurodegenerative diseases and their ability to repress acetylation of core histone proteins, further investigation into the effects of posttranslational modification of histone H1 may be considered important and warranted.50 The myriad of protein−protein interactions ascribed to wildtype Htt confirm its importance as a master regulator of cellular dynamics. In the brain, Htt has been associated with anchoring receptors in the postsynaptic density, retrograde and anterograde axonal transport, neurogenesis, and transcriptional regulation.51 Results presented here single out some of the major protein players that potentially underwrite these pathological outcomes. Radixin (RDX; Table 5), is the only member of the ezrin, radixin moesin (ERM) protein family responsible for the anchoring of GABAA receptor α5 subunits to the cytoskeleton.4 GABAA receptor α5 subunits are preferentially located on MSN of the indirect pathway−the cell type most affected during HD. These cells provide a tonic conductance that, if blocked, reduces the firing rate of MSN.52 A significant reduction in radixin may cause inappropriate motor activity by specifically changing the firing potential of MSN as a result of the loss of extrasynaptic GABAA receptor α5 subunit localization. This spurious protein trafficking may well contribute to the brain region selectivity observed in HD neurodegeneration.53 Memory deficiencies characteristic of HD patients and the altered neuronal morphology observed here in HD-affected neural cells could be a consequence of reduction of IQGAP1 (Table 5), a protein instrumental in dendritic spine construction, neurite outgrowth, and memory formation. Losses of talin, α-actinin, and profilin1 (Table 5) are indicative of cells with impaired motility and migration and may contribute to the inability of newly formed neural progenitors to traffic properly in HD. Because of the limiting number of neurodifferentiated cells available, no other discovery approaches were undertaken in this study. The data obtained, however, now means that the expression of particular markers can be tested for concordance with the proteomics findings through orthogonal immunological approaches. Our data implies that understanding mutant Htt (polyQ expansion) expression impacts on mitochondrial localization and function would provide a cornerstone explanation for the complex HD phenotype. In this regard, the impact of mutant Htt on mitochondrial function is currently the subject of intense investigation,16,54 given the ongoing implications that mutant Htt leads to protein misfolding and aggregation and, importantly, aberrant protein−protein interactions. In their recent study, Ratovitski et al.16 used tandem affinity purification and quantitative proteomics (iTRAQ) to compare and quantify interactions of normal or expanded Htt in striatal cell lines. They found that proteins significantly enriched as “interactors” of expanded Htt were related to “energy production, protein trafficking, RNA posttranscriptional modifications and cell death”. Of the IPA-generated list of interactor proteins found to be more abundant under expanded Htt expression, 15 were found in the IPA-generated list of proteins with significant p values and fold changes in the current study (SI Table S5). Six (ACACA, ATP5B, ATP5F1, SHPD1, IMMT, and RPS7) are directly involved with mitochondrial localization and function, and four (AIFM1, ATP5A1, HSPE1, and TIMM50) are related to mitochondrial-induced cell death. One in particular, AIFM1, with dual functions of energy production and apoptosis, was

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ASSOCIATED CONTENT

* Supporting Information S

(S1) List of identified proteins from undifferentiated pluripotent and neurodifferentiated hESC. The list includes the number of peptides identified from each replicate, fold changes, p values and Ensembl and Uniprot identifiers. (S2) Ingenuity Pathway Analysis Canonical pathways based on proteins with significant p values and fold changes. (S3) Significantly reduced components of the citric acid cycle found in HD-affected hESC. (S4) Observed fold changes in dynamin-like and mitochondrial fission and fusion protein expressions. (S5) The huntingtin (Htt) “interactor” proteins identified in the current study with function, Ensembl protein ID, fold changes, and p values. This material is available free of charge via the Internet at http://pubs.acs.org

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AUTHOR INFORMATION

Corresponding Author

*Phone: +61 2 9850 8211. Fax: +61 2 9850 8313. E-mail: mark. [email protected]. Author Contributions ⊥

L.R.M., H.A.S., M.S.B. and U.S. contributed equally to experimental conceptualization, design, and coordination. M.S.B. and U.S. are equal senior authors. Notes

The authors declare no competing financial interest. J



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ACKNOWLEDGMENTS We thank statisticians D. Pascovici (APAF) and G. Luscombe (Genea) for their assistance with data analysis and statistics and the donation of embryos for research by consenting patients of Genea. Some of the research described herein was facilitated by access to the Australian Proteome Analysis Facility (APAF) established under the Australian Government’s National Collaborative Research Infrastructure Strategy (NCRIS). We thank Dylan Xavier at APAF for assistance with mass spectrometry.

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Proteomics of Huntington's Disease-Affected Human Embryonic

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