Whole gene expression profile in blood reveals multiple pathways deregulation in R6/2 mouse model
© Diamanti et al.; licensee BioMed Central Ltd. 2013
Received: 8 August 2013
Accepted: 15 October 2013
Published: 23 October 2013
Huntington Disease (HD) is a progressive neurological disorder, with pathological manifestations in brain areas and in periphery caused by the ubiquitous expression of mutant Huntingtin protein. Transcriptional dysregulation is considered a key molecular mechanism responsible of HD pathogenesis but, although numerous studies investigated mRNA alterations in HD, so far none evaluated a whole gene expression profile in blood of R6/2 mouse model.
To discover novel pathogenic mechanisms and potential peripheral biomarkers useful to monitor disease progression or drug efficacy, a microarray study was performed in blood of R6/2 at manifest stage and wild type littermate mice. This approach allowed to propose new peripheral molecular processes involved in HD and to suggest different panels of candidate biomarkers. Among the discovered deregulated processes, we focused on specific ones: complement and coagulation cascades, PPAR signaling, cardiac muscle contraction, and dilated cardiomyopathy pathways. Selected genes derived from these pathways were additionally investigated in other accessible tissues to validate these matrices as source of biomarkers, and in brain, to link central and peripheral disease manifestations.
Our findings validated the skeletal muscle as suitable source to investigate peripheral transcriptional alterations in HD and supported the hypothesis that immunological alteration may contribute to neurological degeneration. Moreover, the identification of altered signaling in mouse blood enforce R6/2 transgenic mouse as a powerful HD model while suggesting novel disease biomarkers for pre-clinical investigation.
Huntington disease (HD) is an autosomal dominant neurodegenerative disorder characterized by progressive atrophy of specific brain areas with consequent alterations of motor and cognitive functions, including psychiatric disturbances, weight loss, as well as metabolic, neuroendocrine and immunological alterations [1–3]. The cause of this fatal disease is an aberrant expansion of CAG trinucleotide in the exon 1 of HTT gene, translating into a polyglutamine tract (polyQ) at the N-terminus, and conferring gain-of-function and loss-of-function to wild type huntingtin protein. In healthy individuals, CAG number is included in the range of 6 to 26, while HD patients have more than 36 Gln stretch  with an inverse relationship between polyQ length and the age of onset. The aberrant polyQ tract results in Huntingtin protein misfolding, which generates insoluble intracellular inclusions and aggregates, important hallmarks of the disease.
Despite extraordinary efforts in understanding the HD pathogenesis, the exact molecular mechanisms responsible for this devastating disorder are still unknown and probably multiple parallel processes may contribute, representing potential therapeutic targets [4, 5]. Striking evidences support an important role of transcriptional abnormalities in HD pathogenesis . Mutant Huntingtin protein (mut-HTT) and its aggregates could modulate gene transcription by entering the nucleus and altering the transcriptional machinery either directly, through DNA binding , or sequestering important transcription factors .
Even though no animal models currently in use encompass all disease features, transgenic mice carrying either full length or mut-HTT fragments represent an invaluable instrument to study pathological mechanisms and to test efficacy and toxicity of small molecules in pre-clinical settings [9–11]. Numerous behavioral and neurological symptoms similar to what seen in HD patients have been also observed in transgenic mouse models [11, 12] including those expressing only short fragments of mut-HTT, as R6/2 mice . R6/2 is one of the first HD transgenic mouse model created, expressing only the N-terminal fragment of HTT (exon 1) and it is characterized by short survival and development of pathological features mimicking human stages of disease [13, 14]. For this reason, transcriptional and behavioral alterations have been intensely studied in R6/2, which has being commonly employed not only in pre-clinical drug testing but also in peripheral investigations, often realized in parallel with HD patients [15, 16]. Stemming from those observations, although HD is mainly a CNS disorder, ubiquitous expression of both normal and mut-HTT in the whole body  has moved the attention toward peripheral dysfunctions, increasing the knowledge on HD etiology and other disease manifestations, and aiding the finding of new biomarkers [18, 19], as evidenced by the flourishing number of transcriptional and proteomic studies conducted in HD blood [16, 20–24].
In this study, we investigated transcriptional dysregulations occurring in R6/2 and wild type littermate mouse blood to reinforce previous observations derived from human studies and to discover novel pathological processes and candidate biomarkers, useful in pre-clinical investigations and in designing therapeutic trials. The transcriptional alterations detected in blood were also analyzed in brain to identify a possible link with central pathological mechanisms, and in other peripheral tissues, like skeletal muscle and skin, to validate them as additional sources of potential biomarkers.
Primary analysis of microarrays
GSEA analysis of differentially expressed genes
In order to highlight relevant processes that could be altered in blood, we applied Gene Set Enrichment Analysis (GSEA) method on the same dataset. This computational method identifies groups of genes with modest but coordinate changes in the expression between phenotypes by utilizing all transcripts of a microarray dataset and discovering significant differences in terms of pathways or gene sets associated with a particular biological function. Microarray analysis was performed using KEGG, BioCarta and Reactome pathway collections of MSigDB and was enforced by Biological Processes of GeneOntology.
GSEA canonical pathways results on three different database (KEGG, Reactome and BioCarta)
Gene set name
COMPLEMENT AND COAGULATION CASCADES
PPAR SIGNALING PATHWAY
GLYCINE SERINE AND THREONINE METABOLISM
PRIMARY BILE ACID BIOSYNTHESIS
SYSTEMIC LUPUS ERYTHEMATOSUS
PENTOSE PHOSPHATE PATHWAY
CYSTEINE AND METHIONINE METABOLISM
BIOSYNTHESIS OF UNSATURATED FATTY ACIDS
ONE CARBON POOL BY FOLATE
SELENOAMINO ACID METABOLISM
FATTY ACID METABOLISM
AMINOACYL TRNA BIOSYNTHESIS
CARDIAC MUSCLE CONTRACTION
FORMATION OF FIBRIN CLOT CLOTTING CASCADE
BILE ACID AND BILE SALT METABOLISM
RESPONSE TO ELEVATED PLATELET CYTOSOLIC CA2
CHYLOMICRON MEDIATED LIPID TRANSPORT
METABOLISM OF AMINO ACIDS AND DERIVATIVES
LIPID DIGESTION MOBILIZATION AND TRANSPORT
SYNTHESIS OF BILE ACIDS AND BILE SALTS
SULFUR AMINO ACID METABOLISM
PEPTIDE CHAIN ELONGATION
CLEAVAGE OF GROWING TRANSCRIPT IN THE TERMINATION REGION
TRANSCRIPTIONAL ACTIVITY OF SMAD2 SMAD3 SMAD4 HETEROTRIMER
Similar findings were obtained by analyzing entire microarray expression dataset in terms of biological processes of GeneOntology collections. In particular, 20 of these differentially modulated processes were linked to metabolism (Additional file 1: Table S1). Results confirmed alteration of metabolic processes in mutant mice as well as coagulation impairment and amino acid, lipid and fatty acid metabolism dysfunctions. Moreover, regulation of muscle and heart contraction processes emerged among enriched processes in WT mice.
To understand information derived from GSEA, we analyzed in details pathways markedly modulated in KEGG database, focusing on the two top enriched collections in R6/2, namely “complement and coagulation cascade” and “PPAR signaling”, together with the two top enriched collections in WT mice, that are “cardiac muscle contraction” and “dilated cardiomyopathy”, emphasizing existing relationships with HD pathology.
Complement and coagulation cascade
The complement system is involved in the first line of host defense against pathogens . Consisting of soluble and membrane embedded proteins, it is a component of the innate immune response, that acts in peripheral tissues and in the brain. Although a protective role linked to clearance of cellular debris has been assigned to several components of the cascade, a relevant effect of its activation in brain is that it may contribute to neuro-inflammation and neuronal loss in neurodegenerative disorders as HD and Alzheimer’s disease . Indeed, an increased number of works debate on the immune system activation in HD [2, 27]. Most of them are focused on the complement cascade impairment by accurate investigations at transcriptional and proteomic level, both in blood and brain, to discover potential biomarkers or causative factors of the pathology, respectively [19, 23, 28]. Here, we discovered for the first time transcriptional alteration of numerous components of the complement system in whole blood of an HD mouse model at manifest stage. Although previous transcriptional studies conducted in HD patients blood [20, 21] did not report abnormalities of this pathway, our findings agree with a multi-approaches proteomic investigation performed in HD human plasma samples . In Dalrymple et al. , several proteins including some complement components, as C9, were identified as possible disease progression biomarkers, confirming the involvement of acute-phase response and consequent activation of complement cascade in HD patients. In our investigation, no significant alteration of interleukins and cytokines mRNAs was observed in R6/2 blood at this stage, differently from what has been measured in plasma of HD patients and serum of HD animal models [19, 29].
The identification of complement and coagulation cascade signaling in murine blood may enforce R6/2 transgenic mouse as HD model to discover pharmacodynamic and disease peripheral biomarkers useful in pre-clinical investigation. Moreover, recent evidences focusing on druggability of pathway components [30, 31], evaluated complement factors as potential targets for the treatment of autoimmune and inflammatory diseases, including neurodegenerative disorders.
Considering KEGG database results, PPAR signaling was the second best enriched pathway in mutant mice. This is intrinsically linked to other downstream processes emerging in the KEGG list of R6/2 up-regulated pathways, such as primary bile acid biosynthesis, biosynthesis of unsaturated fatty acids and fatty acid metabolism, or other pathways related to amino acid metabolism or synthesis (Table 1). Additionally, most of them resulted modulated in the analysis performed by Reactome database (Table 1). A similar metabolic profile was also described in serum of HD patients and in an HD murine model .
The other metabolic processes that emerged as altered in R6/2 blood are well-known for being deregulated in HD. Indeed, we found lipid-linked signaling like fatty acids, bile acids and cholesterol metabolism. Altered cholesterol pathway and its biosynthesis were deeply investigated by Cattaneo and collaborators  in patients and animal models, both in peripheral tissues and in CNS , demonstrating that cholesterol might have a pivotal role in HD pathogenesis. Moreover, cholesterol dysfunction is closely associated with lipid, triglyceride and fatty acid metabolism that are also impaired in HD . In conclusion, evidences of PPAR signaling impairment in R6/2 blood, mirroring numerous HD systemic metabolic abnormalities , may contribute to the identification of new pathogenic mechanisms. Drugs against lipid dysregulation have been already tested in HD and they should be supported by disease biomarkers as PPAR target genes, to monitor therapeutic efficacy.
Cardiac muscle contraction and dilated cardiomyopathy
The two processes found down-regulated in R6/2 blood are related to heart contraction performance, which is monitored by cardiac myocytes excitation with the maintenance of cellular homeostasis through Na+ and Ca2+ fluxes. Ca2+ homeostasis imbalance is considered a relevant cause of many neurodegenerative disorders, including HD [37, 38], where impairment of Ca2+ physiological equilibrium is generally followed by progressive heart failure and sudden cardiac death. Additionally, Ca2+ handling defects may exacerbate mitochondrial dysfunctions [39, 40], considered being a relevant mechanism in HD pathogenesis , and hence contributing to cardiac failure too .
A fascinating remark is that, even if our data were generated from a whole genome expression analysis performed in blood, modulation of these two pathways suggests evidences of cardiac dysfunction in R6/2 mice. Nevertheless, although Huntington’s disease is a neurological disorder, cardiac failure is an important cause of death in a high percentage of HD patients (over 20%) . In addition, a recent work showed direct evidences of cardiac dysfunction in R6/2 , highlighting an in-vivo impairment of numerous cardiac functions, including myocardial contractility. Our finding agree with what has been previously demonstrated by Mihm et al. , about cardiotoxic effects coming from peripheral mut-HTT expression. This study displayed that mut-HTT expression in R6/2 cardiomyocytes could result in severe cardiac systolic and diastolic impairment, showing parallel changes between cardiac and neurological symptoms, and suggesting the use of cardiovascular performance indicators as “state biomarkers” of disease progression. Here, we identified for the first time in HD blood a set of deregulated genes involved in cardiac patho-physiology that should be useful to monitor a drug therapy addressing this HD symptom. Peripheral blood gene expression profiling has been already applied to assess some cardiovascular diseases, as myocardial infarction  relying on the ability of blood to operate as a sensor able to capture physiological and pathological modifications, transforming them in gene expression changes .
Selected genes investigated by RT-qPCR
R6/2 vs WT FC
R6/2 vs WT p
tropomyosin 2, beta
solute carrier family 8 (sodium/calcium exchanger), member 1
complement component 3
serine (or cysteine) peptidase inhibitor, clade G, member 1
solute carrier family 27 (fatty acid transporter), member 2
fatty acid binding protein 1, liver
In conclusion, all these findings enforce the hypothesis that immunological alteration may contribute to neurological degeneration and components of this pathway may represent potential biomarkers to monitor in parallel drug efficacy in periphery and in brain, supporting the usefulness of this model in pre-clinical studies.
HD is a neurological and devastating disorder with an urgent need of effective therapies and well established biomarkers. Even though major efforts in finding peripheral disease progression biomarkers are pursued in human studies, the availability of validated animal disease models is of great aid in all pre-clinical studies aimed at moving ahead promising compounds into clinical studies, evaluating their possible molecular mechanism of action. In addition, transcriptional analysis on these models could unveil novel therapeutic targets related to the pathology onset and progression.
On the other side, one of the major drawbacks for in-vivo pathology models is the incomplete correspondence of these with the real human disease situation that could cause misinterpretation of data, with deleterious effects on drug discovery progression. Here, we evaluated through a whole genome approach, peripheral transcriptional alterations of biological pathways in circulating blood cells of a well characterized HD mouse model. Our data revealed the direct involvement of processes that are well known markers of Huntington’s disease progression in man. Several microarray findings have been investigated by RT-qPCR in other peripheral easily accessible tissues and in brain with obvious projection toward clinical studies. The approach adopted in this work offers interesting remarks that could be further investigated in a larger cohort of transgenic mice at different stages of pathology development and validated in other HD models to strengthen the findings on the involvement in HD progression of these biological pathways obtained by GSEA analysis. Nevertheless, preliminary results presented here could be considered a further step in validating the R6/2 mouse model as first line pre-clinical tool, giving at the same time an insight of important pathological hallmarks for Huntington’s disease, and contributing to attractive novel biomarkers development.
Mouse handling, blood and tissues collection
This study included four R6/2 male mice (about 110 CAG repeats) and four wild type (WT) male littermates that were bred at PsychoGenics Laboratories (Tarrytown, NY, USA). All animals were examined, manipulated, and weighed prior initiation of the study to assure adequate health, suitability and to minimize non-specific stress associated with manipulation. During the course of the study animals were maintained in a 12/12 light/dark cycles with temperature ranging between 20 and 23°C and relative humidity around 50%. Chow and water were provided ad libitum for the duration of the study. Genotype was determined by PCR analysis of DNA tail, performed at PsychoGenics Laboratories.
Mice were sacrificed at the age of 16 weeks. Terminal whole blood samples were collected via closed cardiac puncture on anesthetized live mouse (CO2). Blood samples were drawn by sterile syringe and 100 μl were directly transferred to RNAprotect Animal Blood Tubes (BD QIAGEN cat n° 76544) according to the manufacturer indications and maintained at −80°C. In addition, the following organs were collected: brain with cerebellum, skeletal muscle (quadriceps) and skin (a strip of about 1 cm × 3 cm of the abdominal area).
All experiments on mice were carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Institutional Animal Care and Use Committee of PsychoGenics, Inc. (PHS OLAW animal welfare assurance number A4471-01), an AAALAC International accredited institution (Unit #001213).
Total RNA was purified from whole blood by RNeasy Protect animal blood kit (QIAGEN GmbH, Hilden, Germany) according to the instruction provided with the kit. Purified RNA was quantified by Nanodrop ND-1000 Spectrophotometer and checked for RNA integrity by Bioanalyzer (Agilent Technologies, Palo Alto, CA). All samples with RIN between 8.40 and 9.0 were included in the subsequent investigations: RNA obtained from blood of a wild type mouse did not satisfy quality control and was excluded from transcriptional investigations (RIN = 7.50).
Total RNA was extracted from brain (ensuring to dissect exactly the same portion, including striatum and cortex), skeletal muscle, and skin after shaving using RNeasy Lipid Tissue Mini kit (QIAGEN) following the instruction provided by the kit.
To assess RNA integrity (28S and 18S ribosomal RNA bands) all samples were loaded on agarose gel with Ethidium Bromide (Sigma-Aldrich Corporation, MO, USA) and quantified by NANODROP 2000 spectrophotometer (Thermo Fischer Scientific, MA, USA).
Gene expression analysis
Microarray experiment was performed on individual blood samples, 500 ng of total RNA of each sample was employed to generate target RNA or amplified RNA (aRNA). This aRNA was then hybridized onto GeneChip® Mouse Genome 430 2.0. This array comprises over 45,000 probe sets representing more than 34,000 well-substantiated mouse genes. Microarrays experiment was performed by Precision Biomarker Resources, Inc. (Evanston, IL, USA).
Before cDNA synthesis, the same quantity of RNAs derived from every blood sample of each strain was pooled together obtaining a single pooled sample of wild type mice blood and a single pooled sample of mutant mice blood. The two deriving pools were quantified by NANODROP 2000 Spectrophotometer (Thermo Fischer Scientific, MA, USA). cDNA was synthesized from 1 μg of RNA obtained from blood pooled samples as well as from all the other individual tissues (brain, skeletal muscle and skin), by using QuantiTect Reverse Transcription kit (QIAGEN GmbH, Hilden, Germany) and diluted to 100 μL.
RT-qPCRs on cDNA samples were performed using a CFX96 Real Time System/C1000 Thermal cycler (Bio-Rad Hercules, CA, USA) with iQ™ SYBR Green master mix 2X (Bio-Rad) with primers that specifically amplified target or control genes. The run protocol used in RT-qPCR was the “2 step amplification”, with annealing temperature tailored on the primer pair used. Each run was followed by Melting Curve run and analysis to confirm amplification of a single product.
Complete primer sequences and gene bank accession numbers of selected genes analyzed in RT-qPCR
Primer sequence Forward
Primer sequence Reverse
Bioinformatics and statistical analyses
Affymetrix CEL files were analyzed with R program (The R Project for Statistical Computing http://www.r-project.org/) by making use of Bioconductor packages (http://www.bioconductor.org). Normalization for quantitative comparison of different microarrays was performed with quantiles within the RMA package [56–58] and final expression values were calculated by summarizing probe set intensities with median-Polish algorithm. Differential expression analysis between four mutant and three wild type samples was tested with LIMMA package . Correction for multiple testing was not performed due to the low number of arrays.
In RT-qPCR, relative expressions, obtained from Cq corrected by amplification efficiency, were normalized on geometric mean of selected reference gene expression values .
Differentially modulated genes in brain, skin and muscle among mutant and wild type samples were identified with Student’s t-test on logarithmic transformed expressions, p-values below 0.05 were considered statistically significant. No statistical test was applied on blood samples, because these samples were pooled in RT-qPCR investigations. Statistical analysis of RT-qPCR data was performed in GraphPad Prism 5.
Gene set enrichment analysis of microarray data was performed using GSEA 2.07 software ( , http://www.broadinstitute.org/gsea/index.jsp) and using MSigDB v 3.1, focusing attention on the following collections: C2 BioCarta gene sets, C2 KEGG gene sets, C2 Reactome gene sets and C5 GO Biological Processes. Software default parameters were used, except for permutation type parameter that was set to “permutation on gene set” due to the low number of arrays employed. Gene sets with false discovery rate (FDR) less than 0.25 were considered significant.
KEGG pathways were downloaded and processed in R/Bioconductor by using Pathview package .
This work has been supported by European Union - PADDINGTON project, contract n. HEALTH-F2-2010-261358. (http://www.paddingtonproject.eu/).
We thank Giovanna Tripepi and Enrica Diodato for PADDINGTON project management and Carla Scali for guidance in animal models.
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