Identification of differentially expressed genes profiles in a combined mouse model of Parkinsonism and colitis

Different cellular mechanisms have been described as being potentially involved in the progression of neurodegeneration in Parkinson’s disease, although their role is still unclear. The present study aimed to identify in detail, through differentially expressed genes analysis by bioinformatics approaches, the molecular mechanisms triggered after a systemic insult in parkinsonian mice. To address this objective, we combined a dextran sodium sulfate (DSS)-induced ulcerative colitis experimental mice model with an acute 1-methyl-4-phenyl-1,2,3,6-tetradropyridine (MPTP) intoxication. The animals were divided into four experimental groups based on the different treatments: (i) control, (ii) DSS, (iii) MPTP and (iv) MPTP + DSS. The data obtained by microarray and functional enrichment analysis point out the implication of different molecular mechanisms depending on the experimental condition. We see, in the striatum of animals intoxicated only with DSS, dysfunction processes related to the blood. On the other hand, oxidative stress processes are more prominent at the MPTP intoxicated mice. Finally, differentially expressed genes within the MPTP + DSS show functional enrichment in inflammation and programmed cell death. Interestingly, we identify a significant synergistic negative effect of both toxins since the expression of differentially expressed genes (DEGs) related to balanced cellular homeostasis was not enough to prevent processes associated with cell death. This work provides detailed insights into the involvement of systemic inflammation, triggered after an insult in the colon, in the progression of the degeneration in Parkinsonism. In this way, we will be able to identify promising therapeutic targets that prevent the contribution of inflammatory processes in the progression of Parkinson’s disease.

www.nature.com/scientificreports/ To clarify the implication of systemic inflammation in PD, different combinations of experimental animal models have been reported. The most commonly used animal model to approach this issue is based on the systemic administration of lipopolysaccharide (LPS). Extensive research has shown that this endotoxin, from gram-negative bacteria, activates microglia and produces a progressive and cumulative loss of dopaminergic neurons over time 4 . For example, Qin et al. reported that LPS stimulates cells in the liver to produce TNF-α that is distributed in the blood to the brain to induce the synthesis of more TNF-α and, consequently, damaging dopaminergic neurons 5 . In another study, the systemic administration of LPS was combined with the induction of ulcerative colitis by the oral ingestion of dextran sulphate sodium (DSS). The results from this work showed an exacerbation of LPS-induced damage in the nigrostriatal system 6 . Furthermore, García-Domínguez and colleagues combined LPS with the 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP)-based model of PD. The obtained data reinforces the activation of microglial related-events together with the exacerbation of www.nature.com/scientificreports/ the dopaminergic neurodegeneration 7 . Together with these observations, in our previous study, we showed an exacerbation of the dopaminergic neuronal death and the glial activation both in the SNpc and striatum when combining MPTP and DSS intoxications 8 . Despite the published literature, further analyses are yet required to elucidate in depth the cellular and molecular mechanisms triggered after a systemic insult in Parkinsonian mice. In the present study, we performed microarray and bioinformatics analysis to identify differentially expressed genes (DEGs) of mice treated with MPTP and/or DSS. This work's aim was to provide a better understanding of the effect of systemic inflammation on the development of PD.

Regimen of intoxication for DSS and MPTP. Ulcerative colitis intoxication was induced for 8 days by
oral administration of 2-2.5% of DSS (molecular weight, 36-50 kDa, MP Biomedicals LLC, OH, USA) in tap water. On day fourth, MPTP + HCl was intraperitoneal administrated with two injections at 2-h intervals in 1 day dissolved in 0.9% saline (15 mg/Kg, Sigma Aldrich, St Quentin). Mice were distributed in four experimental groups: (a) Control (n = 4), (b) DSS (n = 4), (c) MPTP (n = 5) and (d) MPTP + DSS (n = 5) (Fig. 1a). On day 8, mice were sacrificed by decapitation and brains were immediately removed and dissected into striatum and midbrain 8 . Samples were correctly stored depending on their future use.
RNA extraction and purification for microarray analysis. Striatums were homogenated using QIAzol from the miRNeasy Mini Kit (Qiagen, Hilden, Germany) and stored at -80 °C until RNA extraction. Total RNA was extracted using the miRNeasy Mini Kit according to the manufacturer's instructions. RNA samples were quantitated on a NanoDrop 2000 (Thermo Fisher Scientific, Whaltham, MA). RNA quality was examined on an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Palo Alto, CA) using the RNA 6000 Nano Kit. RNA integrity numbers (RINs) of the samples ranged between 9.2 and 9.7. Samples were stored at -80 °C until microarray experiments.  Figure 3. Summary of the main GO terms found for biological process in the comparisons for (a) DSS vs control and (b) MPTP vs control. The color of the circles help to better identify the groups of terms for specific processes: green for immune system process, pink for response to toxic substance, blue for apoptotic pathway and purple for response to unfolded protein. Analysis of the array expression. The raw data obtained was analyzed with the statistical language R 9 and limma package was used 10 . Intra-array normalization was performed with the method of Loess and inter-array normalization with the Aquantil method. The expression levels of the signals belonging to the same gene were that brings together enrichments from several databases, those of our interest were the annotations from Gene Ontology (GO) and Reactome. From the obtained GO annotations, we carefully summarize and remove redundant terms using Revigo 12 . These results were illustrated in a network diagram using Cytoscape 13 for each ontology for comparisons: biological process (BP), molecular function (MF) and cellular component (CC) (Fig. 1b). The relationships between the nodes are based on the hierarchical association between the terms of the ontology. The brightness of the nodes refers to the level of significance.
Ethics approval and consent to participate. Not applicable.

Results
Differential gene expression profiles. Firstly, normalization from the raw data ( Fig. 2a.i) was made within ( Fig. 2a.ii) and between groups ( Fig. 2a.iii). To avoid any supervised analysis, volcano plots from all the comparisons between the experimental groups showed the distributions of the intensities (Fig. 2b). Moreover, samples from the four experimental groups were distinctly separated in the principal component analysis (PCA) plot, indicating a differential gene expression profile caused by both intoxications (Fig. 2c).
Overall, a total of 2,413 genes were found to be differentially expressed, with an absolute fold change set at > 2 and p-value < 0.05 in all possible comparisons between the experimental groups [see Supplementary file 1]. Of the total DEGs, different number of genes were specifically identified based on the experimental group: DSS (132 genes), MPTP (345 genes) and MPTP + DSS (1,357 genes) compared with the control group, and MPTP compared with MPTP + DSS (281 genes) suggesting different effects in the striatum (Table 1). Moreover, in the independent hierarchical clustering represented as heat-maps, it was confirmed the DEGs within and between groups [see Supplementary file 1], specifically exacerbated in the comparisons between MPTP + DSS and control groups.
GO enrichment analysis. Based on the high amount of differential expressed genes between groups, we aimed to perform a functional enrichment analysis using g:GOSt from gProfiler which provides the most www.nature.com/scientificreports/ enriched GO terms together with other data sources (data shown in the Supplementary Files) associated with our gene lists. Specifically, the data from the GO enrichment analysis is organized into three categories: (i) biological process, (ii) cellular component and (iii) molecular function. All the GO terms lists obtained in each category were summarize removing redundant terms using REVIGO. The output data was used to generate the GO-terms network graphs in Cytoscape. The setting threshold for the false discovery rate (FDR at 0.05).
Biological process. Gene ontology enrichment analysis revealed different profiles depending on the experimental groups. Specifically, the results that concern biological process mainly involved GO terms for response to toxic substance/stimulus, detoxification and immune system but in different way depending on the experimental groups. Thus, up-regulated DEGs, related to response to toxic substance/stimulus, were found in groups treated with DSS (DSS and MPTP vs control; and, MPTP + DSS vs MPTP, Figs. 3 and 4). Interestingly, we found up-regulated DEGs related to detoxification mechanisms in animals treated with DSS compared to control mice ( Table 2, Fig. 3a) and MPTP + DSS, clearly observed when comparing MPTP + DSS vs MPTP (Table 3, Fig. 4).
Moreover, the data revealed that genes related to immune system processes are up-regulated in DSS (Fig. 3a), MPTP (Fig. 3b) and MPTP + DSS (Fig. 4a) groups compared to the control group; and, when comparing MPTP + DSS with MPTP group (Fig. 4b). However, these genes are especially exacerbated when comparing www.nature.com/scientificreports/ MPTP + DSS with control and MPTP mice (Fig. 4a.ii and Table 3). Together with the exacerbation of the immune system process, we also observed in this group oxidative stress processes (Fig. 4a.iii and Table 3), cell activation and cell death (Fig. 4a.iii and Table 3).  (Fig. 5). Interestingly, in the three comparisons (animals treated with DSS) the groups of genes related to haptoblobin-hemoglobin complex and the extracellular region stand out. In addition, when animals are treated together with MPTP ( Fig. 5b and c.i, Table 4), changes are also observed in the intracellular part.

Molecular function. Regarding molecular function GO annotations, we found terms from up-regulated
DEGs mainly implicated in antioxidant activity and haptoglobin binding in the comparisons for DSS vs control, MPTP + DSS vs control and MPTP + DSS vs MPTP (Fig. 6, Table 5).

Reactome enriched analysis.
The data obtained from the Reactome annotations divided the comparisons into different events in the same scenario (striatum level). Firstly, DSS compared to control stand out mechanisms related to heme dysfunction (as scavenging of heme from plasma or heme biosynthesis) possibly due to the bleeding produced by the DSS administration. Since free heme promotes the conversion of low density lipoproteins into cytotoxic products, make it toxic, it is also observed immune system processes up-regulation (Table 6).
Regarding MPTP compared to control, we observed two main pathways: (i) programmed cell death by apoptosis induced DNA fragmentation, and (ii) cellular responses to external stimuli that implicated DNA damage/ telomerase stress induced senescence (Table 6).
On the other hand, the last events are exacerbated when comparing MPTP + DSS with both control and MPTP mice, which are the immune system processes. Specifically, it is observed: (i) the innate immune system response, with the up-regulation of the neutrophil degranulation and the toll-like receptor cascade (regulation of TLR by endogenous ligand) and, (ii) the adaptive immune system response, with the Class I MHC mediated antigen processing and presentation (antigen processing cross presentation) and immunoregulatory interactions between a lymphoid and a non-lymphoid cell ( Table 6). www.nature.com/scientificreports/

Discussion
Prior work have remarked the involvement of systemic inflammation in the development of PD. The findings from the present work extend those published reports through a bioinformatics approach. We aimed to elucidate in depth the molecular and cellular mechanisms triggered after a systemic insult in the striatum of mice untreated and treated with MPTP. The importance of specifically studying the effect of both toxins in the striatum is based on previous data obtained by immunohistochemical techniques in which we observed exacerbation of the inflammatory events when combining both toxins 8 . The results from this study provide novel information about the DEGs in the striatum of mice intoxicated only with DSS. It is clear to observe the up-regulation of processes related to detoxification and inflammatory mechanisms (Fig. 3, Table 3) mainly triggered by the bleeding and lesion of the mucosal surface caused by the administration of DSS 14 . This is the first study to our knowledge to describe the molecular and cellular mechanisms activated in the striatum after a local insult in the colon.
Otherwise, when comparing MPTP mice with the control group, we found biological processes related to the response to unfolded protein (Fig. 3b, Table 2). Interestingly, these results are in accordance with the observations of Fornai and colleagues who described that the administration of MPTP reduces the protein degradation function of the striatal ubiquitin-proteasome system 15 . Later on, it was added that this could lead the accumulation of unfolded proteins and consequently, activates stress-induced cell death mechanisms 16 . Moreover, it was demonstrated that oxidative stress processes may participate in the formation of cross-linked protein aggregates 17 . In these animals, we also noticed DEGs associated with an attempt to regulate negatively the apoptotic signalling pathway and positively the activation of the inflammatory response (Table 3). Interestingly, the data from Reactome analysis showed annotations related to the execution of apoptosis mechanisms induced by DNA fragmentation and the induction of cellular senescence. These results are in line with those reported to another study that suggests that the nature and severity of DNA damage may determine the cellular response 18 . More research is needed to clarify how is the interplay of both routes after a DNA insult 19 . www.nature.com/scientificreports/ Most notably were the observations that resulted from the comparison between MPTP + DSS and control animals. As this work indicates, we obtained an exacerbation of the up-regulated DEGs in all the analysis related to detoxification, oxidative stress and immune system processes.
Our results not only reinforce those results reported in the literature but also extend detailed information on the contribution of systemic inflammation to the progression of the neurodegeneration associated with Parkinson's disease. Although our aim was highly achieved, we are aware of some intrinsic limitations such as the number of animals used in this type of studies 20,21 or the focus in one brain area. The relevance of this lies in the contribution of novel and detailed information that describes different expression profiles that can be used as a guide for further and specific analysis. We advise that future work should evaluate upregulated pathways in different brain areas over time. In this way, we will be able to identify promising therapeutic targets that prevent the contribution of inflammatory processes in the progression of Parkinson's disease.

Conclusions
Altogether, we provide functional and comprehensive bioinformatics analyses of the deleterious effect of the systemic inflammation in the striatum of MPTP intoxicated mice. Interestingly, the data showed in this study describes a scenario that becomes more complex when combining both treatments. Thus, the processes related www.nature.com/scientificreports/ www.nature.com/scientificreports/ to inflammation and oxidative stress are exacerbated resulting in a significant up-regulation of the cell death mechanisms.

Data availability
All data generated or analysed during this study are included in this published article [and its supplementary information files].