A Comprehensive Diverse ‘-omics’ Approach to Better Understanding the Molecular Pathomechanisms of Down Syndrome

Diverse ‘-omics’ technologies permit the comprehensive quantitative profiling of a variety of biological molecules. Comparative ‘-omics’ analyses, such as transcriptomics and proteomics, are powerful and useful tools for unraveling the molecular pathomechanisms of various diseases. As enhanced oxidative stress has been demonstrated in humans and mice with Down syndrome (DS), a redox proteomic analysis is useful for understanding how enhanced oxidative stress aggravates the state of individuals with oxidative stress-related disorders. In this review, ‘-omics’ analyses in humans with DS and mouse models of DS are summarized, and the molecular dissection of this syndrome is discussed.


Introduction
Understanding the changes in molecules provides important and valuable information for evaluating the molecular pathomechanisms of diseases. Diverse '-omics' technologies permit the quantitative monitoring of various biological molecules in a comprehensive manner. Comprehensive expression profiles are designated by adding the suffix '-omics' onto previously used terms, such as prote-omics (protein), transcript-omics (transcripts), lipid-omics (lipid), metabol-omics (metabolites), and so on ( Figure 1). Comparative '-omics' analyses comparing the profiles of healthy subjects and populations with some illness are a powerful and useful approach for better understanding molecular pathomechanisms. Indeed, a variety of disorders, including neurological disorders, have been subjected to '-omics' analyses.
Although most '-omics' analyses assess the levels of target molecules, protein, transcripts, fatty acids, and various metabolites in organs, redox proteomics and phospho-proteomics indicate the degree of oxidative stress and activation of signal transduction or transcriptional factors, respectively. Thus, proteomics can be used to determine not only the protein expression, but also the degree of post-translational modifications, such as phosphorylation and oxidation. However, no single '-omics' analysis can fully unravel the complexities of some disorders. Therefore, the integration of multiple layers of information-the multi-'-omics' approach-is required to acquire a precise picture of the pathophysiology of a given disease.
carrying three copies of various segments of MMU16 syntenic to HSA21 have been recently established: Dp1Tyb, Dp2Tyb, Dp3Tyb, Dp4Tyb, Dp5Tyb, Dp6Tyb, and Dp9Tyb [7] (Figure 2). The Ts65Dn model is the most intensively studied model of DS to date and carries a 13.2 Mb segmental trisomy of MMU16, and the Ts1Cje model carrying a shorter segmental trisomy (7.6 Mb) is also widely used. Both models display DS-like phenotypes, including learning and memory deficits and/or an abnormal neuron morphology and function [3,4,[8][9][10].  In this review, we discuss the changes in the molecules in brains from individuals with Down syndrome (DS) and DS mouse models according to '-omics' analyses. Although DS is caused by trisomy of human chromosome 21 (HSA21), the distal end of mouse chromosome 16 (MMU16) and part of MMU10 and MMU17 share synteny with human chromosome 21 (HSA21) [1] (Figure 2). Therefore, several mouse models carrying an extra partial MMU16, MMU10, or MMU17 have been established as mouse models for DS ( Figure 2). In particular, MMU16 has synteny with a large portion of HSA21, which has led to the establishment of several mouse models of DS: Dp(16)Yey [2], Ts65Dn [3], Ts1Cje [4], Ms1Ts65 [5], and Ts1Rhr mice [6] (Figure 2). Furthermore, seven mouse models of DS carrying three copies of various segments of MMU16 syntenic to HSA21 have been recently established: Dp1Tyb, Dp2Tyb, Dp3Tyb, Dp4Tyb, Dp5Tyb, Dp6Tyb, and Dp9Tyb [7] (Figure 2). The Ts65Dn model is the most intensively studied model of DS to date and carries a 13.2 Mb segmental trisomy of MMU16, and the Ts1Cje model carrying a shorter segmental trisomy (7.6 Mb) is also widely used. Both models display DS-like phenotypes, including learning and memory deficits and/or an abnormal neuron morphology and function [3,4,[8][9][10].

Comparative Transcriptomics in the DS Brain
The analysis of the expression of transcripts in humans with DS and mouse models of DS provides valuable information for understanding the pathophysiological mechanisms underlying this disease. However, whether or not the expression of trisomic genes is upregulated in a gene-dosedependent manner and the effect of trisomic gene expression on the expression of transcripts from euploid genes remain unclear.
A number of comprehensive gene expression analyses using tissue of humans with DS and mouse models of DS have been performed. The findings from studies using brain tissue are summarized in Table 1. Although relatively few studies have been performed with human DS samples, two included in Table 1 indicate that a gene-dose-dependent increase in transcription was detected in the brain both before and after birth [11,12].
A number of comparative transcriptomic analyses in brains of mouse models of DS have been performed ( Table 1). The precise brain region and the age of the samples analyzed must be considered for an accurate interpretation of these comparative transcriptomics data. However, a variety of samples have been used, and most (but not all) transcriptomic analyses comparing trisomic and euploid tissues support the hypothesis of increased mRNA levels in a gene-dose-dependent manner. In addition, a number of reports have shown that the mRNA expression of some euploid genes is disturbed in the brain of DS model mice.

Comparative Transcriptomics in the DS Brain
The analysis of the expression of transcripts in humans with DS and mouse models of DS provides valuable information for understanding the pathophysiological mechanisms underlying this disease. However, whether or not the expression of trisomic genes is upregulated in a gene-dose-dependent manner and the effect of trisomic gene expression on the expression of transcripts from euploid genes remain unclear.
A number of comprehensive gene expression analyses using tissue of humans with DS and mouse models of DS have been performed. The findings from studies using brain tissue are summarized in Table 1. Although relatively few studies have been performed with human DS samples, two included in Table 1 indicate that a gene-dose-dependent increase in transcription was detected in the brain both before and after birth [11,12].
A number of comparative transcriptomic analyses in brains of mouse models of DS have been performed ( Table 1). The precise brain region and the age of the samples analyzed must be considered for an accurate interpretation of these comparative transcriptomics data. However, a variety of samples have been used, and most (but not all) transcriptomic analyses comparing trisomic and euploid tissues support the hypothesis of increased mRNA levels in a gene-dose-dependent manner. In addition, a number of reports have shown that the mRNA expression of some euploid genes is disturbed in the brain of DS model mice.  Males at postnatal day 0 Affymetrix murine genome U74A and U74B microarrays The expression of most genes in the trisomic region was increased approximately 1.5-fold, and the top 24 most consistently over-expressed genes in Ts1Cje mice were all located in the trisomic region.
In the cerebellum of Ts1Cje mice, six homeobox genes and two genes belonging to the Notch pathway showed severely decreased expression [20] Ts1Cje mice (B6C3SnF1/ Orl) The expression ratios of 54% of trisomic genes (Ts1Cje/WT) were significantly higher than the expected diploid gene ratio of 1.0.
Ts1Cje neural progenitors proliferated at a slower rate. Some euploid genes involved in proliferation, differentiation, and the glial function were dysregulated. [24] Brain Sci. 2017, 7, 44 6 of 14 Although transcriptomic analyses using DS brains in humans and mice provide important information, as described above, the interpretation of the data is very complex. RNA sequencing is a powerful analytical tool that is expected to replace microarrays for transcriptomics profiling [25]. RNA sequencing has several advantages over microarrays, such as the avoidance cross-hybridization, the limited detection range of individual probes, and non-specific hybridization. RNA sequencing could, therefore, provide new information on the gene expression in brains with DS.

Comparative Proteomics in the DS Brain
The protein expression does not always correlate with the expression of mRNA, so a comprehensive analysis of the protein expression in brains with DS is very important for understanding the pathomechanisms of the disease. A number of comparative proteomics analyses using the brains from patients and mouse models have been performed. The reports including such analyses for understanding changes in the protein expression in the brains with DS and neuronal cells derived from embryonic stem (ES) cells with an extra HSA21 are listed in Table 2. In contrast to the findings on gene expression analyses, a number of proteomics analyses failed to detect any increased expression of trisomic genes at the protein level (Table 2). This might be due to the sensitivity of the proteomic analysis, especially two-dimensional (2D)-electrophoresis, since antibody arrays successfully detected an increase in the protein expression of trisomic genes [26]. Recently-developed chemical labeling techniques using isobaric tags (i.e., tags with the same molecular weight) for the relative and absolute quantification (iTRAQ) [27] and tandem mass tags (TMT) [28] are expected to resolve the issue of the low sensitivity of separation on 2D-electrophoresis.
A number of proteins associated with proteolysis, energy metabolism, the cytoskeleton, and cell proliferation that are coded in disomic genes were found to be dysregulated in humans and mice with DS (Table 2) [29][30][31], although proteins, which were differentially expressed in both human and mouse brain with DS, have not yet been identified in these proteomic analysis. For instance, ubiquitin carboxyl-terminal hydrolase isozyme L1 (Uchl1) coded on an euploid gene showed a decreased expression in the brain of a 141G6 mouse carrying a yeast artificial chromosome (YAC), which contains HSA21 genes, Down syndrome critical region gene 3 (Dscr3), phosphatidylinositol glycan anchor biosynthesis, class P (Pigp), ripply transcriptional repressor 3 (Ripply3), and tetratricopeptide repeat domain 3 (Ttc3), as well as neurons differentiated from ES cells carrying an extra HSA21 [31,32]. In addition, proteolysis and energy metabolism-related proteins, such as Uchl1 and α-enolase, show not only differential expression, but also oxidative modification under DS conditions, as will be explain in the next section.

Redox Proteomics in the DS Brain
Since proteins are major targets of reactive oxygen, nitrogen species and aldehyde products of lipid peroxidation (LPO), comparative analysis of these protein modifications reveals the degree of oxidative stress, and the certain target molecules of enhanced oxidative stress. Therefore, redox proteomics is a useful tool to decipher the effect of oxidative stress in the brain with DS. As shown in Table 3, redox proteomics detecting carbomylated-or 4-hydroxynonenal (4-HNE) adducted-proteins revealed that the carbonylated UchL1 level was increased in the human DS brain [33]. Furthermore, although the levels of LPO-related modified proteins, such as 13(S)-hydroperoxyoctadeca-9Z,11E-dienoic acid (13-HPODE)-adducted α-enolase, glycolytic enzyme, and 4-HNE-adducted neuron specific enolase, are increased in Ts1Cje mice [34], the expression of α-enolase and neuron-specific enolase decreased in the human DS cortex and the hippocampus of 141G6 mice [29,32]. The impairment of the energy metabolism is suggested to be closely related to several age-related neurodegenerative disorders, such as Alzheimer's disease and mild cognitive impairment (MCI) [35]. Thus, the oxidative stress-mediated decline in certain protein functions may be associated with abnormalities of the DS brain. Only a small number of trisomic proteins were increased in a gene-dose-dependent manner.
Ts65Dn mice have lost the correlations seen in control mice among levels of functionally related proteins, including the components of the MAP kinase pathway and subunits of the NMDA receptor.

Other '-omics' Analyses
Other '-omics' analyses, such as metabolomics including lipidomics and elementomics, including metallomics, are expected to prove useful in understanding the pathophysiological state of DS. As no proteins were found to be differentially expressed in the Ts1Cje mouse brain during neonatal and postnatal life by 2D-electrophoresis-based proteomics [30], these other '-omics' approaches are thought to be particularly useful.
A metabolomics approach can digest a very large volume of information regarding the levels of the complete set of low-molecular-weight molecules or metabolites synthesized by a cell. The alteration of biogenic metabolites, such as neurotransmitters and prostaglandins, in abundance provides a unique chemical fingerprint that specific cellular processes leave behind. Although there have been a number of metabolomics studies on the accumulation of such elements in the brains of Alzheimer's disease patients (see [44]), no metabolomics studies with DS subjects have yet been reported.
Elementomics, including metallomics, is another new '-omics' approach using inductively-coupled plasma-mass spectrometry (ICP-MS) [45]. Elementomics comprehensively elucidate the levels of the most common elements, including biogenic trace metals, in the tissues and cells. This approach has only been applied to study Alzheimer's disease, with no reports in DS subjects available at present [46,47]. The dyshomeostasis of intrinsic metals, such as zinc, copper, and iron, may play a role in cognitive impairment in individuals with DS, as intersectin 1 coded in the trisomic region in DS is suggested to be involved in iron internalization. Indeed, increases in the levels of non-protein-bound iron in serum and iron-binding protein lactotransferrin in the brain have been demonstrated in individuals with DS [48,49]. Other '-omics' analyses, such as metabolomic and elementomics analyses, for DS samples are expected to help unravel the pathophysiological mechanisms underlying abnormalities in the DS brain.