Knock-Down of Heterogeneous Nuclear Ribonucleoprotein A1 Results in Neurite Damage, Altered Stress Granule Biology, and Cellular Toxicity in Differentiated Neuronal Cells

Abstract

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is an RNA binding protein (RBP) that is localized within neurons and plays crucial roles in RNA metabolism. Its importance in neuronal functioning is underscored from the study of its pathogenic features in many neurodegenerative diseases where neuronal hnRNP A1 is mislocalized from the nucleus to the cytoplasm resulting in loss of hnRNP A1 function. Here, we model hnRNP A1 loss-of-function by siRNA-mediated knock-down in differentiated Neuro-2a cells. Through RNA sequencing (RNA-seq) followed by gene ontology (GO) analyses, we show that hnRNP A1 is involved in important biological processes, including RNA metabolism, neuronal function, neuronal morphology, neuronal viability, and stress granule (SG) formation. We further confirmed several of these roles by showing that hnRNP A1 knock-down results in a reduction of neurite outgrowth, increase in cell cytotoxicity and changes in SG formation. In summary, these findings indicate that hnRNP A1 loss-of-function contributes to neuronal dysfunction and cell death and implicates hnRNP A1 dysfunction in the pathogenesis of neurodegenerative diseases.

Keywords: Neuro-2a cell line; RNA binding protein; heterogeneous nuclear ribonucleoprotein A1; neurodegenerative disease; neuronal cell damage; small interfering RNA.

Figure 1.

Efficient knock-down of hnRNP A1 in differentiated Neuro-2a cells. A, Undifferentiated Neuro-2a cells were treated with four different siA1 duplex oligonucleotides for 72 h, which showed varying degrees of hnRNP A1 knock-down. B, Quantification of A demonstrating siA1#4 was the most potent siA1 duplex oligonucleotide to significantly decrease hnRNP A1 expression compared with siNEG. Unpaired t test (ns = non-significant, *p < 0.05, **p < 0.01); n = 3 biological replicates. Data are plotted as mean ± SEM. C, Neuro-2a transfection, differentiation and data collection protocol. D, Protein from differentiated Neuro-2a cells treated with either siNEG or siA1 for 72 h were assayed by Western blotting for hnRNP A1 and β-actin. E, Band densitometry of Western blottings as in D demonstrates a significant decrease in hnRNP A1 protein expression after 72 h of treatment with siA1 as compared with siNEG. Unpaired t test (***p < 0.001); n = 3 biological replicates. Data are plotted as mean ± SEM. F, Confirmation of decreased hnRNP A1 expression (green) following treatment with siA1 using immunocytochemistry. Scale bar: 20 μm. G, Corrected total hnRNP A1 nuclear fluorescence was measured using ImageJ. Cells in the siA1 condition demonstrated significant reduction in hnRNP A1 expression as compared with siNEG-treated cells. Unpaired t test (****p < 0.0001); n = 3 biological replicates. Individual cell values (n = 30 cells per replicate) are plotted as mean ± SEM.

Figure 2.

RNA-seq analysis of hnRNP A1 knock-down in differentiated Neuro-2a cells. A, PCA analysis of log transformed normalized RNA-seq data showing that siA1 and siNEG formed distinct clusters with strong intercluster separation. B, Heatmap of DE transcripts plotted as normalized count values for siNEG-treated (n = 3) and siA1-treated (n = 3) cells. C, Volcano plot of siA1-treated samples (siA1 vs siNEG) illustrating significantly upregulated (green dots) and downregulated (red dots) transcripts. Non-DE transcripts are represented as black dots; p threshold of 0.05 is displayed in gray. See Extended Data Figure 2-1 for list of significant DE genes. D–G, GO enrichment analysis of DE genes identified GO terms related to RNA metabolism (D), neuronal functions (E), neuronal morphology (F), cell death (G), and RNP complex (H). Values at the end of each bar represent number of DE genes in each GO process. Data are presented as -log₁₀false discovery rate (FDR) values, which represent p-values adjusted for multiple tests by Benjamini–Hochberg procedure. See Extended Data Figure 2-2 for list of significantly enriched GO terms from biological processes.

Figure 3.

HnRNP A1 binding to DE genes. A, Pie chart representing the subset of DE genes with human orthologs (n = 1341) that had previously been shown to be known hnRNP A1 binding targets (89.86%) and those that had not (10.14%). B, Pie chart representing subset of upregulated DE genes with human orthologs (n = 729) that had previously been shown to be known hnRNP A1 binding targets (88.89%) and those that had not (11.11%). C, Pie chart representing subset of downregulated DE genes with human orthologs (n = 612) that had previously been shown to be known hnRNP A1 binding targets (91.01%) and those that had not (8.89%).

Figure 4.

Effect of hnRNP A1 knock-down on neuronal health. A, Immunofluorescent images of Neuro-2a cells stained for DAPI (blue), hnRNP A1 (green), and β-III-tubulin (red) to identify neurites. Cells in the siNEG condition have more neurites that appear longer as compared with the siA1 condition. Scale bar: 20 μm. B, Neurites were traced in the β-III-tubulin channel in ImageJ using the NeuronJ plugin as described in Materials and Methods. Quantification revealed that siA1-treated Neuro-2a cells have significantly fewer neurite branches as compared with the siNEG condition. Unpaired t test (***p < 0.001); n = 3 biological replicates. Individual cell values (n = 30 cells per replicate for siNEG; n = 20 cells with >50% knock-down for siA1) are plotted as the mean ± SEM. C, Corrected total hnRNP A1 nuclear fluorescence of Neuro-2a cells treated with siA1 correlates with neurite branch number. PC test (r = 0.167, r² = 0.02,799, p = 0.0498); n = 3 biological replicates. Individual cell values (n = 30 cells per replicate) are plotted. D, Neuro-2a cells treated with siA1 have significantly shorter neurites as compared with the siNEG condition. Unpaired t test (***p < 0.001); n = 3 biological replicates. Individual cell values (n = 30 cells per replicate for siNEG; n = 20 cells with >50% knock-down for siA1) are plotted as mean ± SEM. E, Corrected total hnRNP A1 nuclear fluorescence of Neuro-2a cells treated with siA1 correlated with neurite sum length. PC test (r = 0.2959, r² = 0.08,758, p = 0.0015); n = 3 biological replicates. Individual cell values (n = 30 cells per replicate) are plotted. F, HnRNP A1 knock-down significantly increased cellular cytotoxicity as compared with siNEG-treated cells as measured by the CYQUANT LDH cytotoxicity assay. Unpaired t test (*p < 0.05); n = 3 biological replicates. Data are plotted as mean ± SEM.

Figure 5.

HnRNP A1 knock-down affects SG formation. A, Immunofluorescent images of differentiated Neuro-2a cells treated with siNEG or siA1 for 72 h followed by 30-min treatment with sodium arsenite. Cells are stained for β-III-tubulin (blue), hnRNP A1 (red), and G3BP (green) to identify SGs. Cells in the siNEG condition have significantly more punctate-like G3BP⁺ granules as compared with the siA1 condition. Scale bar: 20 μm. B, Quantification revealed that sodium arsenite-treated Neuro-2a cells in the siA1 condition form significantly fewer SGs as compared with the siNEG condition. Unpaired t test (****p < 0.0001); n = 3 biological replicates. Individual cell values (n = 90 cells per replicate for siNEG; n = 141 cells with >50% knock-down for siA1) are plotted as mean ± SEM. C, HnRNP A1 cell fluorescence of Neuro-2a cells treated with siA1 followed by sodium arsenite treatment correlates with number of SGs. PC test (r = 0.2825, r² = 0.07,982, p < 0.0001); n = 3 biological replicates. Individual cell values (n = 90 cells per replicate) are plotted. D, Quantification revealed that sodium arsenite-treated Neuro-2a cells in the siA1 condition have significantly smaller SGs as compared with the siNEG condition. Unpaired t test (****p < 0.0001); n = 3 biological replicates. Individual cell values (n = 90 cells per replicate for siNEG; n = 141 cells with >50% knock-down for siA1) are plotted as mean ± SEM.