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A transcriptomic characterization of immortalized hippocampal ts16 cell lines to further elucidate the hippocampal dysfunction in Down Syndrome

Thesis by Carrie Hicks

Advisor: Dr. Daniel Paredes

Abstract

Down Syndrome (DS, trisomy 21, T21) is caused by a triplication of human chromosome 21. The genetic disease affects roughly 1 in 700 births. While the majority of DS phenotypes vary in presence and severity by individual, all T21 individuals experience some level of cognitive disability that primarily impacts learning and memory processes. The ability of synapses, communication sites between neurons, to strengthen or weaken over time is known as synaptic plasticity. Synaptic plasticity allows for the brain to learn and store information. This process is dependent on the correct branching and growth of axons and dendrites in response to environmental cues. The trisomy 16 (Ts16) mouse model exhibits DS associated features but rarely survives gestation. The link between the hippocampus and synaptic plasticity has been extensively studied because the hippocampus is an important brain structure for learning and memory processes. The present study aims to characterize the transcriptome of an immortalized Ts16 hippocampal cell line by comparing it to that of a WT cell line. The identified differentially expressed genes largely suggest that there is a dysregulation of protein encoding genes regarding actin filament organization and regulation, lamellipodium function, and cell migration in the Ts16 cell line that ultimately prevents correct axonal and dendritic expansion in the hippocampus.

Methods

1. Cell Culture

2. RNA Extraction

3. RNA Sequencing

4. Data Analysis

Methods

Cell Culture

Hippocampal immortalized Ts16 (Htk) and WT (H1b) cell lines were obtained from the Caviedes research group, Faculty of Medicine, University of Chile, Santiago, Chile (Cárdenas et al. 2002). Htk and H1b cells were plated into 60mm TPP dishes in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham supplemented with 2.5% Fetal Bovine Serum, 10% Adult Bovine Serum, and 0.2% Normocin (Invivogen). The media was exchanged for Neurobasal Medium (Thermo Fisher) supplemented with 2% B27, 0.1% bFGF-2 Recombinant Protein 100 ng/mL (Thermo Fisher), 0.3% GlutaMAX (Thermo Fisher), 0.1% Normocin (Invivogen), and 10 µM Forskolin. The cells were allowed to differentiate for 72 hours

Cell Culture

RNA Extraction

Total RNA was extracted using TRIzol reagent (Sigma) according to manufacturer’s protocol. Total RNA from TRIzol precipitation was loaded onto RNA spin columns (Machery Nagel) and DNaseI treated on column, and further purified according to manufacturer’s protocol. Initial RNA concentration and purity was assessed via NanoDrop One (Thermo Scientific) and frozen at -80°C. RNA quality and assessment and RNA seq was performed by The Genomics and Microarray Core Facility. cDNA Libraries were prepared from mRNA and 150bp paired end read with sequencing was performed employing the Illumina HiSEQ4000 at 20million read depth.

RNA Extraction

Immortalized hippocampal T16 cell lines after hours of growth (A), 72 hours differentiation (B) and 96 hours of differentiation (C). RNA was extracted after 96 hours of differentiation.

RNA Sequencing

RNA-seq sequences, provided in FASTQ format by the Genomics and Microarray Core Facility at the University of Colorado, Denver, were aligned using salmon version 1.0.0 (https://github.com/COMBINE-lab/salmon/releases) and Ensembl98 Mus musculus genome sequence and annotation (https://uswest.ensembl.org/info/data/ftp/index.html). Differential gene expression was performed using DESeq2, an R/Bioconductor package for differential analysis of count data (https://www.bioconductor.org/packages/release/bioc/html/DESeq2.html) (Patro 2017; Love 2014)

RNA Sequencing

Data Analysis

Analysis

ClueGO is a Cytoscape app that can extract representative functional biological information for large lists of genes or proteins (Mlecnik, 2018). ClueGO analyses were performed using Cytoscape 3.7.2 and ClueGO 2.5.5. Analysis was done similar to Mazzarino et al., (2019) with differences highlighted. The GO and Reactome releases were Mus musculus_GO-EBI-UniProt-GOA_27.02.2019 and Mus musculus_REACTOME_27.02.2019. Differentially expressed gene (DEG) lists used for ClueGO were curated against ClueGO databases for coding genes ensuring recognition of gene abbreviation entries. The top 150 most positively and 150 most negatively changed gene (Top 300 DEGs) were used as input lists. ClueGO analyses were performed against the following databases: Gene Ontology biological processz, Gene Ontology cellular component, Gene Ontology molecular function, KEGG, Reactome pathways, and Reactome reactions. Analyses were performed using default parameters, evidence code set to “all” network specificity set to “representative” with a 3 gene/term cutoff and visualization set to groups. These setting approximate terms found in gene ontology levels 3-11 and color code terms based upon their associated parental groupings.

Results

We used ClueGO to further investigate the pathways and mechanisms that are comprimised in the trisomic cells. RNAseq revelealed differentially expressed genes (DEGs) among the trisomic (Htk) and disomic (H1b) hippocampal cells. These genes were analyzed by the three gene ontologies, the Reactome Knowledgebase, and KEGG to produce the following results.

Results

Gene Ontologies

Gene ontologies are organized into three categories: biological processes, cellular component, and molecular function. Genes that are referred to in the category of biological processes are those that contribute to the completion of a specific biological objective. Genes sorted into the cellular component gene ontology are those that refer to product localization. The molecular function ontology refers to the biochemical activity of the gene products

Biological Processes

Genes that are referred to in the category of biological processes are those that contribute to the completion of a specific biological objective. Differentially expressed genes (DEGs) between the disomic and trisomic hippocampal Ts16 cells mapped to 210 shared terms within 44 groups.

Biological Processes

Differentially Expressed Genes

Groups and Genes in the Biological Processes Gene Ontology related to forebrain neuronal development. Information was found using the Entrez and GeneCards summaries on GeneCards

Molecular Functions

Molecular Function

The molecular function ontology refers to the biochemical activity of the gene products

The molecular function ontology contains 39 shared terms across 17 groups. The most notable of this ontology include exopeptidase activity (15.38%), regulation of voltage gated calcium channel activity (12.82%), serine type endopeptidase activity (10.26%) heme binding (10.26%) and regulation of cation channel activity (7.69%). Other groups of relevance to the MF ontology include dynein intermediate chain binding, MHC Class II protein complex binding, and aspartic type peptidase activity

Differentially Expressed Genes

Groups and Genes in the Molecular Function Gene Ontology related to forebrain neuronal development. Information was found using the Entrez and Gene Cards summaries on the Gene Cards website.

Cellular Components

The cellular component ontology produced 24 shared terms across 13 groups. The most notable of this ontology include Lamellipodium with 20.83%, extrinsic component of the plasma membrane with 16.67%, and MHC Class II Protein Complex (12.5%)

Cellular Components

Groups and Genes in the Cellular Components Gene Ontology related to forebrain neuronal development. Information was found using the Entrez and GeneCards summaries on GeneCards.

Differentially Expressed Genes

Reactome Knowledgebase

Reactome Knowledgebase

The Reactome Knowledgebase is organized into two categories: reactions and pathways. Reactions are the basic unit of the knowledgebase and an organism's complete set of reactions is known as the Reactome.

Reactome Reactions

The results produced by Reactome reactions contains 56 shared terms across 9 groups. Secretion of collagens is the largest group of the ontology by far, containing 50% of the shared terms. CSNK1D phosphorylates SEC23 and assembly of anterograde IFT train are also notable groups, with 17.86% and 10.71% of the shared terms, respectively.

Reactome Reactions

Groups and Genes in Reactome Reaction related to forebrain neuronal development. Information was found using the Entrez and GeneCards summaries on GeneCards.

Differentially Expressed Genes

Reactome Pathways

The Reactome Pathway results contained 44 shared terms across 10 groups. Terms regarding extracellular matrix organization are the most common among the results, containing 43.18% of the terms. G beta:gamma signaling through BTK contains 15.91% and arachidonic acid metabolism contains 1.36%. ECM proteoglycans, COP II mediated vesicle transport, and muscle contraction are also notable groups to the dataset. The network map revealed one overlap between extracellular matrix organization and ECM proteoglycans groups

ReactomePathways

Groups and Genes in the Reactome Pathways related to forebrain neuronal development. Information was found using the Entrez and GeneCards summaries on GeneCards.

Differentially Expressed Genes

KEGG

The KEGG ontology contains 34 shared terms across 14 groups. Inflammatory bowel disease, ovarian steroidogenesis, and Ras signaling pathway made up the largest groups containing 32.26%, 12.9% and 12.9% of the shared terms. Primary immunodeficiency, axon guidance and amoebiasis also contained notable numbers of shared terms

KEGG

Groups and Genes in the KEGG results related to forebrain neuronal development. Information was found using the Entrez and Gene Cards summaries on Gene Cards website

Differentially Expressed Genes

Discussion

This study aimed to conduct a transcriptomic analysis on an immortalized hippocampal Ts16 cell line to further elucidate the biological mechanisms behind hippocampal dysfunction in the DS model. Current literature has established the link between intellectual disability, a universal symptom of DS, and altered neural and dendritic spine morphology and quantity in the hippocampus (Becker, 1991). The establishment of Ts16 immortalized cell lines provides a platform for an examination of the molecular mechanisms that result in Ts16, and potentially T21, phenotypes. The transcriptomes of WT and Ts16 immortalized hippocampal cell lines were compared using ClueGO, an app supported by Cytoscape. Differentially expressed genes (DEGs) between the mouse models were assigned to shared terms that represented the biological pathway, component or function with which they are involved. The shared terms were organized into groups that represent a larger cell process. This process of organization occurred in using three gene ontologies (biological processes, cellular components, and molecular functions), two Reactome Knowledgebases (Reactome reactions and reactome pathways) and with the KEGG pathway database. The groups are organized into pie charts based on their percentage of the total DEGs in the dataset.

Future Directions

Biological Pathways Analysis

DEG doublecortin (DCX) , a gene found throughout the Biological Processes Gene Ontology, is a protein encoding gene that produces a cytoplasmic protein that is responsible for the regulation, organization and stability of microtubules. Reduced expression of DCX following RNAi transfection has been shown to reducing branching and complexity of dendrites, as a result of higher rates of dendritic pruning and lower rates of dendritic elongation (Cohen, 2007). DCX continues to be expressed during adult neurogenesis in adult proliferating progenitor cells (Cohen, 2007). Llorens- Martin, et al. found that an increase in physical exercise can increase DCX expression in WT mice but not in Ts65Dn (an alternative DS mouse model) demonstrating that the T21 condition likely directly impacts DCX expression (Llorens- Martin, 2010). The molecular mechanism for embryonic neurogenesis by DCX is not currently understood.

DEG Dystrophin (Dmd) (Table 1) produces an actin binding protein that connects the inner cytoskeleton to the ECM. Brain dystrophin is abundant in the hippocampus and absent in other subcortical areas (Lidov, 1993). The absence of dystrophin in mice causes altered calcium homeostasis and hippocampal neuronal function, particularly impacting LTP, and impairs spatial and recognition memory (Vaillend, 2004). Overall, current literature suggests that the dystrophin protein is important for the normal structure and function of synapses. However, like DCX, its molecular mechanism in memory formation is not well established in literature

Cellular Components Analysis

The largest group of DEGs in the Cellular Components GO was Lamellipodium (Figure 4). The lamellipodium is made up of thin and flat membrane structures at the cell periphery. This structure is rich in branched actin filaments and undergoes cycles of protrusion and retraction to push a portion of the cell through its substrate (Small, 2002). DEG Actin Binding LIM Protein 1 (Ablim1) is a LIM zinc-binding protein that binds to actin filaments to mediate interactions between actin and cytoplasmic targets (Table 2). The first isoform of DEG PDZ and LIM Domain 4 (Pdlim4) encodes a protein that binds to alpha-actin-1 (ACTN1) to increase its affinity to bind with F-actin and promote the formation of actin stress fibers. Pdlim4 is known to assist with the direction of the synaptic AMPA receptor in dendritic spines to the post-synaptic neuron. The second isoform of Pdlim4 is involved in the organization of the actin cytoskeleton and regulation of cell migration (Table 2). This suggests that the regulation and organization of the lamellipodium, a critical structure for dendrite expansion, is compromised in the T16 hippocampus. Lamellipodium extension relies on cross-linking of actin filaments and the data above suggests that proteins involved in the actin organization and regulation that allows for the lamellipodium to permeate substrate may be acting improperly, suggesting that the lamellipodium could be a target for future therapies (Stossel, 2001).

The axonal growth cone refers to the larger structure that protrudes from the cell body of a neuron to explore the extracellular environment and determine the appropriate direction of growth (Tanaka, 1995). Gene coding proteins Cob1, Nin, and Tiam1 are DEGs in the cellular components GO and play roles in actin and microtubule regulation and organization (Table 2). DEG Plld produces a component of actin containing filaments.

The molecular functions GO (Figure 5) contains DEG Aebp1, a positive regulator of collagen fibrillogenesis (Table 3). The protein encoded by this gene is responsible for organization and regulation of the ECM. This gene is also significantly altered in the AD mouse hippocampus (Hokama, 2013). Similarly, the largest group of the Reactome Reaction (Figure 6, Table 4) and Pathway (Figure 7, Table 5) databases contains a variety of collagen producing proteins that play large roles in the formation of the extracellular matrix, as well as genes Fn1, Lamb3, Capn6, Fbn1, Matn3, Spta1 and Vcan that play roles within the ECM to facilitate processes such as cell migration and adhesion.

Molecular Function Analysis

Other processes that appear to be dysregulated include neuronal crest development, blood pressure homeostasis, immune system development, and gastrointestinal activity, all of which are associated with T21. For example, the presence of Sema3 (Table 1) and Erg1/2 (Table 6) indicate a dysregulation of neural crest development. The neural crest is comprised of bilaterally paired strips of cells that appear in the ectoderm during embryogenesis and eventually migrate and differentiate to different regions of the embryo. Many systems receive contributions from the neural crest cells, including the head and face (Minoux, 2010). A dysregulation in the neural crest may contribute to abnormal craniofacial features, such as the palpebral fissure observed in many DS patients (Roper, 2009).

Reactome and KEGG Analysis

Conclusions

A comparison of the transcriptomes of WT and T16 immortalized hippocampal cells allows for us to gain insight into the differently expressed proteins that may create some of the phenotypes observed in T16 mice and ultimately T21 individuals. Our data largely supported that the dysfunction of the hippocampus is due to abnormal cell migration and thus dendritic expansion as a result of deregulated actin organizing and binding proteins. Future research should focus on using the most severely dysregulated proteins as targets for future therapies.

Conclusions

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