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. 2020 Aug 13:12:256.
doi: 10.3389/fnagi.2020.00256. eCollection 2020.

Transcriptome Analyses in BV2 Microglial Cells Following Treatment With Amino-Terminal Fragments of Apolipoprotein E

Tanner B Pollock  1 , Giovan N Cholico  1 , Noail F Isho  2 , Ryan J Day  2 , Tarun Suresh  1 , Erica S Stewart  1 , Madyson M McCarthy  1 , Troy T Rohn  1
Affiliations

Transcriptome Analyses in BV2 Microglial Cells Following Treatment With Amino-Terminal Fragments of Apolipoprotein E

Tanner B Pollock et al. Front Aging Neurosci. .

Abstract

Despite the fact that harboring the apolipoprotein E4 (APOE4) allele represents the single greatest risk factor for late-onset Alzheimer's disease (AD), the exact mechanism by which ApoE4 contributes to disease progression remains unknown. Recently, we demonstrated that a 151 amino-terminal fragment of ApoE4 (nApoE41-151) localizes within the nucleus of microglia in the human AD brain and traffics to the nucleus causing toxicity in BV2 microglia cells. In the present study, we examined in detail what genes may be affected following treatment by nApoE41-151. Transcriptome analyses in BV2 microglial cells following sublethal treatment with nApoE41-151 revealed the upregulation of almost 4,000 genes, with 20 of these genes upregulated 182- to 715-fold compared to untreated control cells. The majority of these 20 genes play a role in the immune response and polarization toward microglial M1 activation. As a control, an identical nApoE31-151 fragment that differed by a single amino acid at position 112 (Cys→Arg) was tested and produced a similar albeit lower level of upregulation of an identical set of genes. In this manner, enriched pathways upregulated by nApoE31-151 and nApoE41-151 following exogenous treatment included Toll receptor signaling, chemokine/cytokine signaling and apoptosis signaling. There were unique genes differentially expressed by at least two-fold for either fragment. For nApoE31-151, these included 16 times as many genes, many of which are involved in physiological functions within microglia. For nApoE41-151, on the other hand the number genes uniquely upregulated was significantly lower, with many of the top upregulated genes having unknown functions. Taken together, our results suggest that while nApoE31-151 may serve a more physiological role in microglia, nApoE41-151 may activate genes that contribute to disease inflammation associated with AD. These data support the hypothesis that the link between harboring the APOE4 allele and dementia risk could be enhanced inflammation through activation of microglia.

Keywords: Alzheimer’s disease; BV2 cells; M1 phenotype; RNA-seq; apolipoprotein E4; inflammation; microglia cells; toxicity.

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Figures

FIGURE 1
FIGURE 1
Orthogonal projections of confocal Z-stacks show nuclear localization of amino-terminal fragments of nApoE3 and E4 following exogenous treatment in BV2 microglia cells. BV2 microglia cells were treated with either 25 μg/ml of nApoE31–151 (A–E) or with nApoE41–151 (F–J) for 24 h, fixed, and immunostained with anti-His antibody to detect the localization of the apoE fragments. Confocal images were captured and Z-stacks were constructed showing the subcellular localization of nApoE fragments. (A) High magnification merged image depicting nApoE31–151 in green and DAPI nuclear staining in blue. (B) Inset orthogonal projection from red rectangle in panel A demonstrating nuclear localization of nApoE31–151 in a single BV2 microglia cell (crosshair). (C–E) Low magnification of nApoE31–151 labeling with DAPI (blue, C), nApoE31–151 (green, D), and the overlapped image (E). All scale bars represent 10 μm. (F–J): Identical to panels A–E except staining is representative of nApoE41–151 labeling. As with nApoE31–151, orthogonal images clearly demonstrate the nuclear labeling of nApoE41–151 (crosshair, G). Data are representative of five individual experiments.
FIGURE 2
FIGURE 2
Functional categorization of up- and downregulated genes in BV2 microglia cells treated with an amino-terminal fragment of apoE4. BV2 cells were left untreated or treated for 5 h with nApoE41–151 followed by total RNA purification and transcriptome analyses. The x-axis reflects the number of genes included for each component, process, or function. For example, there were a total of 75 upregulated genes categorized as being a part of the cellular component. Functional characterization was based on gene ontology (GO) annotations. Detailed information is shown in Supplementary Data File S1.
FIGURE 3
FIGURE 3
Validation of transcriptome analysis by RT-PCR and ELISA of two identified upregulated genes. Two genes (CXCL2 and IL12B) identified by transcriptome analyses to be significantly upregulated in BV2 cells following treatment with 25 μg/ml apoE41–151 (Table 1) were independently tested by RT-PCR (A,C) or ELISA (B,D) in order to validate transcriptome findings. (A) BV2 cells were left untreated (control, green bar) or treated for 5 h with 25 μg/ml nApoE41–151 (blue bar), and RNA was extracted. RT-PCR analysis indicated a ∼1,500-fold increase in the expression of the inflammatory chemokine, CXCl2. Data are representative of two independent experiments. (B) Secreted CXCl2 levels are significantly elevated following treatment of BV2 cells with nApoE41–151 (blue bar) as compared to untreated controls (green bar). Data are representative of five independent experiments ± SEM *denotes p-value is <0.0001 between control and nApoE41–151. (C) BV2 cells were left untreated (control, green bar) or treated for 5 h with 25 μg/ml nApoE41–151 (blue bar), and RNA was extracted. RT-PCR analysis indicated a ∼388-fold increase in the expression of the inflammatory cytokine, IL-12b. Data are representative of two independent experiments. (D) Secreted IL-12b levels are significantly elevated following treatment of BV2 cells with nApoE41–151 (blue bar) as compared to untreated controls (green bar). Data are representative of eight independent experiments ± SEM *denotes p-value is < 0.00001 between control and nApoE41–151.
FIGURE 4
FIGURE 4
Enriched biological processes and classification of apoptosis related genes regulated by an amino terminal fragment of nApoE4. BV2 cells were plated onto 6-well plates to confluency and treated with or without ApoE41–151 for 5 h. Following treatment, total RNA was extracted and transcriptome analysis was carried out as described in the Materials and Methods. (A) Data are expressed as fold enrichment of biological processes in BV2 microglia cells in the presence of the ApoE41–151. Up-regulated processes are involved in the inflammatory immune response and the activation of BV2 microglia, while down-regulated processes are involved in cell division. (B) Classification of genes in the apoptosis signaling pathway regulated by ApoE41–151. A total of 66 out of 72 genes in the apoptosis signaling pathway (P00006) were influenced by the introduction of ApoE41–151. Enrichment and classification analyses were conducted using the PANTHER classification system (pantherdb.org).
FIGURE 5
FIGURE 5
Enriched biological processes and classification of apoptosis related genes regulated by an amino terminal fragment of nApoE3. BV2 cells were plated onto 6-well plates to confluency and treated with or without nApoE31–151 for 5 h. Following treatment, total RNA was extracted and transcriptome analyses was carried out as described in the Materials and Methods. (A) Data are expressed as fold enrichment of biological processes in BV2 microglia cells in the presence of the nApoE31–151. Up-regulated processes are involved in the inflammatory immune response of BV2 microglia, while down-regulated processes are involved in mitochondrial oxidative phosphorylation. (B) Classification of genes in the apoptosis signaling pathway regulated by nApoE31–151. Numerous genes involved in cell signaling pathways were upregulated following treatment with the E3 fragment. Enrichment and classification analyses were conducted using the PANTHER classification system (pantherdb.org).
FIGURE 6
FIGURE 6
Gene expression following treatment with fragments displays similar patterns of upregulation. The scatterplot analysis displays a moderate positive relationship between the nApoE31–151 and nApoE41–151 treatment groups (S = 9651720, ρ = 0.537, p-value < 0.00). Data is representative of three independent trials that were averaged and expressed as a percentage change in comparison to the control versus each treatment. A Spearman Rank Correlation was utilized to overcome dissonance in normality from extreme data points. Ellipses represent 0.5 and 0.95 confidence intervals and the dotted line is a linear trendline.
FIGURE 7
FIGURE 7
Venn diagram demonstrating common and divergent gene regulation between nApoE31–151 and nApoE41–151 fragments. A comparison of genes regulated more than two-fold by treatment with nApoE31–151 and nApoE41–151. nApoE31–151 regulated the expression of 1654 genes that were not regulated by nApoE41–151. Of these 1654 genes, 1010 were upregulated and 644 were downregulated. Conversely, there were 102 genes uniquely regulated by nApoE41–51. Of these 102 genes, 81 were upregulated, and 21 were downregulated. There were 1673 genes that were regulated by both nApoE41–151 and nApoE31–151. Also included are common differentially expressed genes (DEGs) for both fragments including 1,062 induced DEGs and 591 repressed DEGs (orange intersecting region).
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