With the advent of mass spectrometry and bioinformatic platforms, high-throughput proteomic studies for different tissues, under various differentiation stages or disease conditions, have proliferated in the literature. Among a few quantitative proteomic techniques, SILAC has recently gained popularity for global-scale analysis of proteins in different cell conditions . One notable advantage of this metabolic labelling technique is that nearly all peptides of all proteins can contribute to quantification, unlike other labelling techniques that target a group of peptides with certain characteristics to be labelled. We hence utilized SILAC to identify differences in the proteome of amniotic fluid cells from T21-affected versus CN fetuses, to identify molecular pathways that are responsible for DS pathogenesis.
The next major step after a large-scale discovery phase is selection of the most promising candidates and verification in individual samples by more elaborate quantification methods. Our initial filtering criteria for selecting candidates were based on differences between the control pair (CN:CN) and the experimental pairs (T21:CN). For example, when we considered proteins with differences exceeding 3 standard deviation in H/L ratios, the control pair showed 38 proteins, whereas the experimental pairs showed 150 to 300 proteins. These findings suggest that a large number of amniocyte proteins are expressed in different amounts between the CN and T21 conditions.
There are at least two reasons as to why our quantification based on SILAC may potentially have a relatively large variability. First, amniocytes in primary culture do not represent a homogenous population, unlike most other cell cultures. It has been observed previously, as well as in the current study, that only a subset of amniocytes survive after a few doubling times and the amniocyte cultures become relatively homogeneous, although the exact nature of these cells are yet to be determined . Second, the amniocytes used in this study originated from different individuals. Therefore, the results were expected to be significantly more variable, compared to studies that use immortalized cells from one individual. Given that proteins that show differential expression in only one experimental pair may be due to analytical variability, only proteins that showed differential expression across two or more experimental pairs from our initial list of 904 proteins were retained for further analysis. Here, we employed SRM assay for verification of SILAC data, since we have previously validated its accuracy and effectiveness for verification of candidates in amniotic fluid .
Network modeling suggested that a number of pathways include multiple proteins that are found in our list of dysregulated proteins (Figure 2). For example, a pathway that includes NF-κB was one of our top 3 pathways, and NF-κB, along with NFATc, has been implicated in the dysregulation of DS candidate region-1 [16, 17]. Another pathway that includes APP was one of our top 3 pathways, and 29 out of the 35 involved proteins of this particular network were identified in our list of 904 proteins that seem to be dysregulated. APP gene encodes a transmembrane protein called amyloid precursor protein in humans, which can be sequentially cleaved by the action of the β and γ secretases, to produce amyloid-beta (Aβ) peptides. APP protein and its peptides seem to contribute to the pathogenesis of DS by both gain of toxic functions and loss of normal biological functions. Aβ42 peptide is the main constituent of amyloid plaques that are a hallmark of Alzheimer’s disease, and recent studies have suggested that the cognitive decline in Alzheimer’s is mediated by reduction of synaptic plasticity attributed to the Aβ plaque formation . Aβ peptides can also cause cerebral amyloid angiopathy, as these peptides aggregate to coat cerebral blood vessels. Plaques indicating amyloid angiopathy have also been observed in DS-affected brains . Although the exact function of APP is unknown, APP seems to play an important role in differentiation or migration processes of neural stem cells. In vitro studies have shown that APP is required for differentiation of neural stem cells, and in vivo, it was shown that neural stem cells cannot migrate or differentiate in an APP-knockout mouse . Our previous study showed that APP expression in amniotic fluid is increased by two-fold in DS-affected pregnancy, as early as the 16th week of gestation . Based on these previous and our current findings, we can hypothesize that APP metabolism is altered at an early stage of fetal development, and its degree of alteration may be one of the most significant, among numerous molecular pathways that are implicated in the development of DS phenotypes.
Several of the candidate proteins have also been directly or indirectly associated with various symptoms of DS in previous studies. The results obtained for SOD1 and NES seem to be particularly consistent. The SOD1 gene is located on chromosome 21 and it encodes for superoxide dismutase, a ubiquitous protein that is involved in the clearance of free radicals produced within cells. Two types of neural pathologies are associated with this protein. First, pathogenic variants of this protein are prone to proteosomal degradation by ubiquitination processes, and such defects have been associated with amyotrophic lateral sclerosis type 1 (ALS1), a neurodegenerative disorder affecting upper and lower motor neurons . Secondly, SOD1 proteins, both wild-type and variants, have a tendency to form fibrillar aggregates, and these aggregates have cytotoxic effects, resulting in neurodegeneration. Increases in SOD1 and APP were studied together, and only when combined, the double transgenic mice showed severe morphological damage . Our results showed that SOD1, unlike other candidates, was consistently upregulated in T21-amniocytes compared to the controls, and this finding supports the traditional gene-dosage hypothesis even at the protein level. The hypothesis predicts increased expression of genes encoded in chromosome 21 , and previous studies at the mRNA level have showed mostly supportive results [24–26].
Unlike SOD1, there is little information available for NES. This protein seems to be down-regulated according to the results of the present study. NES is an intermediate filament protein that has been associated with Creutzfeldt-Jakob syndrome and pathologic neovascularization. It is expressed in various parts of the human body, including brain, eyes, ovaries, skin, and some pathologic tissues such as glioblastoma. NES expression is also strongly observed in stem cells of the central nervous system in the neural tube, and it has been speculated that it has an important role in central nervous system development . Upon terminal neural differentiation, NES is downregulated and replaced by neurofilaments.
Although bioinformatic databases allow easy annotation of candidates for their function, tissue expression, and potentially involved pathways, understanding of their function must be done within the context of the cell type and state of the cells. Since amniocytes represent a relatively heterogeneous population that has not been fully characterized, speculating on each protein function in the amniotic fluid cell proteome should be approached with caution. For example, there may be an array of proteins that have been well-described in fully differentiated cells, although the same proteins may be actively involved in development and/or cellular differentiation during fetal growth. Therefore, information on their developmental functions from bioinformatic repositories may be very limited. Also, expression of proteins in terminally differentiated cells can be quite different from expression in stem cell-like cells. Moreover, gene dosage clearly depends on the biological function of the product of the gene, including enzymes, structural proteins, transcription factors, intracellular signaling molecules, cell surface markers, and receptors.
There are a few limitations of this study, which originate from the nature of the samples. For example, the heterogenous nature of amniotic fluid cells can introduce false-positives into our list of proteins that reflect DS pathogenesis, warranting a verification step. Also, the heterogeneity of the disease phenotypes and the degree of severity make the analyses more difficult. For example, 50 to 60% of DS individuals suffer from congenital cardiac defects, and some of the altered pathways for heart development could or could not be captured in our candidate list, since not all DS fetuses are affected. Even for the universal phenotypes, such as cognitive development, there is a wide range of severity; therefore “signature proteins” for any of the phenotypes could potentially be missing from our list, especially at such an early stage of development.