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. 2024 Mar 14:12:1342741.
doi: 10.3389/fcell.2024.1342741. eCollection 2024.

Stable knockdown of Drp1 improves retinoic acid-BDNF-induced neuronal differentiation through global transcriptomic changes and results in reduced phosphorylation of ERK1/2 independently of DUSP1 and 6

Marvi Ghani  1   2 , Peleg Zohar  1 , Gyula Ujlaki  1   2 , Melinda Tóth  1 , Hailemariam Amsalu  1   2 , Szilárd Póliska  3 , Krisztina Tar  1
Affiliations

Stable knockdown of Drp1 improves retinoic acid-BDNF-induced neuronal differentiation through global transcriptomic changes and results in reduced phosphorylation of ERK1/2 independently of DUSP1 and 6

Marvi Ghani et al. Front Cell Dev Biol. .

Abstract

Background: Dynamin-related protein Drp1 -a major mitochondrial fission protein- is widely distributed in the central nervous system and plays a crucial role in regulating mitochondrial dynamics, specifically mitochondrial fission and the organelle's shaping. Upregulated Drp1 function may contribute to the pathological progression of neurodegenerative diseases by dysregulating mitochondrial fission/ fusion. The study aims to investigate the effects of Drp1 on retinoic acid-BDNF-induced (RA-BDNF) neuronal differentiation and mitochondrial network reorganization in SH-SY5Y neuroblastoma cells. Methods: We generated an SH-SY5Y cell line with stably depleted Drp1 (shDrp1). We applied RNA sequencing and analysis to study changes in gene expression upon stable Drp1 knockdown. We visualized the mitochondria by transmission electron microscopy and used high-content confocal imaging to characterize and analyze cell morphology changes and mitochondrial network reorganization during neuronal differentiation. Results: shDrp1 cells exhibited fused mitochondrial ultrastructure with perinuclear clustering. Stable knockdown of Drp1 resulted in the upregulation of genes involved in nervous system development. High content analysis showed improved neurite outgrowth, segmentation, and extremities in differentiated shDrp1 cells. Neuronal differentiation was associated with a significant reduction in ERK1/2 phosphorylation, and ERK1/2 phosphorylation was independent of the dual specificity phosphatases DUSP1/6 in shDrp1 cells. Differentiated control underwent mitochondrial morphology remodeling, whereas differentiated shDrp1 cells retained the highly fused mitochondria and developed long, elongated structures. The shDrp1 cells responded to specific apoptotic stimuli like control in vitro, suggesting that Drp1 is not a prerequisite for apoptosis in SH-SY5Y cells. Moreover, Drp1 downregulation reduced the formation of toxic mHtt aggregates in vitro. Discussion: Our results indicate that Drp1 silencing enhances RA-BDNF-induced neuronal differentiation by promoting transcriptional and mitochondrial network changes in undifferentiated cells. We also demonstrate that the suppression of Drp1 reduces toxic mHtt aggregate formation in vitro, suggesting protection against neurotoxicity. Thus, Drp1 may be an attractive target for further investigation in future strategies to combat neurodegenerative diseases.

Keywords: DRP1; DUSP1 and DUSP6; ERK1/2; RNA seq; high-content analysis; huntingtin aggregates; in vitro neuronal differentiation; mitochondrial network rearrangement.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Generating a stably depleted human neuroblastoma cell line for Drp1 using lentiviral technology. Using lentiviral technology, DNM1L/Drp1 expression was downregulated in the SH-SY5Y human neuroblastoma cell line. HEK293T cells were transfected with the mixture of packaging-enveloping vectors and the plasmid pGIPZ-GFP containing the shRNA target sequence to produce the virus. Cells stably expressing empty pGIPZ-GFP were used as a control. Transduction of SH-SY5Y neuroblastoma cells was conducted using 8 μg/mL polybrene, and cells were maintained in media containing 1.25 μg/mL puromycin for selection. (A) The qPCR analysis shows successful silencing of DNM1L in shDrp1 cells. The sequences of primer #1 and #2 are listed in Supplementary Table S3. (B) Total cell lysates from control and shDrp1 cells were separated by SDS-PAGE to analyze Drp1 protein levels. Actin was used as the loading control. (C) Representative transmission electron microscopy images of mitochondria in control (upper panel) and shDrp1 cells (lower panel). Cell and mitochondria contours were manually segmented. Only mitochondria with visible and intact internal membranes were considered for consecutive analyses (insets). Mitochondria were displayed using a default shared colormaps option of Amira 3D with 8 distinct colors. Colors are independent of values; their purpose is to show the mitochondria used for further analyses. Amira 3D (version 2022.1; ThermoFischer Scientific) image analysis software was used to analyze the numerical parameters of the segmented structures. The clustering of mitochondria was quantified as a measure of the closest neighbor of each mitochondrion. Mitochondria were color-coded based on their distance from the closest neighbor. Distance values in μm were extracted using the Label Analysis module of Amira 3D. These distance values were displayed using the Colorize by Measure module of Amira 3D, which colored the mitochondria according to the values. Color scales were added using the Colormap Legend module from Amira 3D. A scale bar was added by the software to the upper right corner of each image. Quantitative analyses of average inside mitochondrial length (μm) (D), total inside mitochondrial length (μm) (E), average (F) and total (G) mitochondrial area (μm2), and average mitochondrial distance (μm) (H). Mann-Whitney test was used for statistical analyses. Only p values p < 0.05 are considered statistically significant.
FIGURE 2
FIGURE 2
Drp1 deficiency affects the transcription of functionally relevant genes. (A) RNA-Seq data analysis from three independent experiments confirmed the depletion of DNM1L (gene name for Drp1) mRNA in the shDrp1 knockdown SH-SY5Y cells. (B) Compared to control cells, the differentially expressed genes (DEGs) in shDrp1 cells included 972 upregulated and 782 downregulated genes. Only significant DEGs are presented (Bejamini-Hochberg FDR corrected p-value <0.05). GO analysis of differentially expressed genes between shDrp1 and control cells significantly enriched several nerve system-related pathways (Bonferroni step-down corrected p-value <0.05). Heatmaps represent the expression patterns of genes of neurogenesis GO:0022008 (C), axon development GO:0007409 (D), regulation of synapse organization GO:0050807 (E), and synapse assembly GO:0007416 (F). Heatmaps were generated by a hierarchical clustering algorithm using Ward’s linkage method, and similarity measures were determined by Euclidean distance metric with the StranNGS software. The color range shows the normalized gene expression values on the log2 scale; the histogram represents the distribution of the genes by their normalized values. (G) The Volcano plot was generated by the online application VolcaNoseR2.https://huygens.science.uva.nl/VolcaNoseR2/. The ten most significantly upregulated and downregulated genes of shDrp1 cells are shown.
FIGURE 3
FIGURE 3
shDrp1 cells show increased neuronal differentiation with longer neurite outgrowth and more interconnected neuronal networks. Control and shDrp1 cells were treated with 10 μM RA on day 0 for 3 days (days 0–3) and 50 ng/mL BDNF on day 3 for 3 days (days 3–6) to induce neuronal differentiation. Automated live-cell confocal microscopy was performed on an Opera Phenix High Content Analysis system. A ×40 water objective was used for image acquisition settings. (A) Representative images of differentiated (Day 6) and undifferentiated control and shDrp1 live cells stained with Hoechst 33342 for nuclei and tubulin tracker for neuron-specific β-tubulin III. Images were merged to visualize both stains. Inset images reveal neurite outgrowth and the presence of several branches in differentiated cells. (Scale bar: 50 µm) (B–G) The mRNA levels of marker genes for neuronal differentiation, including SYN1 (Synapsin 1) (B), SNAP25 (Synaptosomal-associated protein 25) (C), TUBB3 (Tubulin beta-3 chain) (D), NES (Nestin) (E), SOX2 (Transcription factor SOX-2) (F) and DDC (Aromatic-L-amino-acid decarboxylase) (G) were measured by qPCR and using the 2−ΔΔCT method in undifferentiated and differentiated control and shDrp1 cells. Data are presented as the mean ± SD of n ≥ 3 separate experiments. Statistical analysis was performed by ordinary One-way ANOVA with multiple comparisons (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001).
FIGURE 4
FIGURE 4
Quantitative analysis demonstrates a significant increase in neurite outgrowth parameters in shDrp1 cells. Automated confocal microscopy analysis of neurite outgrowth in undifferentiated and differentiated (Day 6) control and shDrp1 live cells was performed on an Opera Phenix High Content Analysis system. Live cells were stained with Hoechst 33342 for nuclei and tubulin tracker for neuron-specific β-tubulin III. Quantitative analysis was performed using the built-in “CSIRO neurite Analysis 2” method of the Harmony 4.9 software. (A–H) Defined parameters were analyzed in three independent experiments. Data are presented as the mean ± SD of three independent experiments (n = 3). 39677 differentiated and 39658 undifferentiated shDrp1 cells and 45066 differentiated and 22568 undifferentiated control cells were analyzed. Groups were compared using ordinary One-way ANOVA with multiple comparisons (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, ns indicates not-significant).
FIGURE 5
FIGURE 5
Reduced phosphorylation of ERK1/2 in shDrp1 cells on Days 3 after RA-induced differentiation. Heatmaps represent the expression patterns of genes regulating MAPK cascade GO:0043408 (A) and ERK1 and ERK2 (B) cascade GO:0070371. Heatmaps were generated by a hierarchical clustering algorithm using Ward’s linkage method, and similarity measures were determined by Euclidean distance metric with the StranNGS software. The color range shows the normalized gene expression values on the log2 scale, and the histogram represents the distribution of the genes by their normalized values. The protein- and phosphoprotein levels of members of cellular signaling pathways, including JNK, phospho-JNK (C), Akt, phospho-Akt (D), p38, phospho-p38 (E), MEK1/2, phospho-MEK1/2 (F) and ERK1/2, phospho-ERK1/2 (G) were examined by western blotting in undifferentiated and differentiated (Days 3 and 6) control and shDrp1 cells. Cells were untreated or treated with 10 μM RA for 3 days (0–3) and 50 ng/mL BDNF for the next 3 days (3–6) to achieve neuronal differentiation. Cells were lysed with RIPA buffer, and an equal amount of protein was separated. β-actin was used as an internal loading control. Representative images of western blots are shown. Statistical analyses of the relative protein expressions are shown. Images were taken using a ChemiDoc Imager, and the pixel intensity was quantified and normalized to the internal control, β-actin, using Image Lab software. Data are presented as means ± SD (n = 4). Groups were compared using One-way ANOVA with multiple comparisons. Only significant differences are shown (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001).
FIGURE 6
FIGURE 6
Inhibition of dual specificity phosphatases 1 and 6 does not lead to increased phospho-ERK1/2 in shDrp1 cells. (A) The optimal working concentration of BCI was obtained by the following: Cells were treated with 10 μM RA for 3 days to induce phase 1 differentiation. Control and shDrp1 cells were treated with 0.1 vol% vehicle DMSO or different concentrations of BCI for 24 h on Day 2. Cells were lysed with RIPA buffer, and an equal amount of proteins were separated to examine the level of phospho-ERK1/2. GAPDH was used as the internal loading control. (B) The protein and phosphoprotein levels of ERK1/2 were examined by SDS-PAGE and western blotting in undifferentiated and differentiated (Day 3) control and shDrp1 cells. Cells were treated with DMSO or 10 μM RA for 3 days to induce phase 1 differentiation. Control and shDrp1 cells were treated with vehicle or 1 μM BCI for 24 h on Day 2. Cells were lysed with RIPA buffer, and an equal amount of proteins were separated. β-actin was used as the internal loading control. Representative images of western blots are shown. Statistical analyses of the relative protein expression of ERK1/2 (C) and phospho-ERK1/2 (D) are shown. Images were taken with a ChemiDoc Imager, and the pixel intensity was quantified and normalized to the internal control β-actin using Image Lab software. Data are presented as means values ±SD (n = 4). Groups were compared using ordinary One-way ANOVA with multiple comparisons. Only significant differences are shown (** indicates p < 0.01).
FIGURE 7
FIGURE 7
Mitochondrial network remodeling of control and shDrp1 cells during neuronal differentiation. Cells were treated with 10 μM RA for 3 days (0–3) and 50 ng/mL BDNF for the next 3 days (3–6) to achieve neuronal differentiation. (A) Automated confocal microscopy was performed on an Opera Phenix High Content Analysis system. A ×63 water objective was used for image acquisition settings. Representative images for mitochondrial morphology in undifferentiated and differentiated (Day 6) control and shDrp1 live cells stained with MitoTracker Red CMXRos for mitochondria and Hoechst 33342 for nuclei are shown. (B) Quantification of mitochondrial classes was performed with the built-in Harmony 4.9 and PhenoLogic machine-learning software. Mitochondria were classified as long, elongated tubular, short tubular, round, compact tubular, and fragmented. The number of objects (mitochondrial populations) were counted in undifferentiated and differentiated control and shDrp1 cells and were expressed as percent % of the overall. > 300 cells were analyzed/sample. Statistical analysis was performed by two-way ANOVA with multiple comparisons (** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates not-significant).
FIGURE 8
FIGURE 8
Reduced expression of mitochondrial fusion proteins MFN1 and MFN2 in shDrp1 cells compared to control cells. The protein levels of mitochondrial fission and fusion proteins were examined using anti-DNM1L (A), anti-Opa1 (B), anti-MFN1 (C), and anti-MFN2 (D) antibodies in undifferentiated and differentiated (Days 3 and 6) control and shDrp1 cells. Cells were lysed with RIPA buffer, and an equal amount of protein was separated using SDS-PAGE and analyzed by western blotting. β-actin was used as an internal loading control. Representative images of western blots are shown. Statistical analyses of the relative protein levels are shown. Images were taken using a ChemiDoc Imager, and the pixel intensity was quantified and normalized to the internal control, β-actin, using Image Lab software. Data are presented as mean values ±SD (n ≥ 3). Groups were compared using One-way ANOVA with multiple comparisons (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates not-significant).
FIGURE 9
FIGURE 9
Increased maximal respiration in differentiated shDrp1 cells. Oxygen consumption rate (OCR) was measured in undifferentiated Day 6 differentiated control and shDrp1 cells. (A) The basal OCR was determined 30 min before and after mitochondrial sequential addition of 1.5 µM oligomycin (olig), 1 µM FCCP, and 1 µM rotenone/antimycin A cocktail (Anti/Rot). (B) Calculated parameters of mitochondrial respiration based on measured OCR. Data were normalized to total protein (pmol/min/µg protein). Data were analyzed using Wave Desktop software. Data are presented as means ± SEM of n = 3 independent experiments. Statistical analysis was performed by two-way ANOVA with multiple comparisons. Only significant differences are shown (* indicates p < 0.05, ** indicates p < 0.01).
FIGURE 10
FIGURE 10
Retinoic acid and selective mitochondrial inhibitors induce cell death in Drp1-depleted cells to a similar extent than in control. (A) Cell viability was assessed using the sulphorhodamine B (SRB) assay. Undifferentiated control and shDrp1 cells were treated with 0.1 vol% vehicle DMSO and 10 µM RA. Data are presented as the means ± SD of three independent experiments. Groups were compared using One-way ANOVA with multiple comparisons. (B) The effects of Drp1 depletion on cellular death after differentiation. Cellular death was monitored using propidium iodide (PI) staining in undifferentiated and differentiated control and shDrp1 cells. Results are presented as means ± SD of three independent experiments. Statistical analysis was performed using One-way ANOVA with multiple comparisons (* indicates p < 0.05). (C) Assessment of cell viability using the SRB assay. Undifferentiated control and shDrp1 cells were treated with DMSO, 10 µM Rotenone, 3 µM Oligomycin, or 100 nM Antimycin (A). Data are presented as means ± SD of three independent experiments. Statistical analysis was performed using One-Way ANOVA with multiple comparisons. Only significant differences are shown (* indicates p < 0.05).
FIGURE 11
FIGURE 11
Stable knockdown of Drp1 delays the aggregate formation of mutant N-Htt fragments. Control and shDrp1 cells were transfected with pHM6-Q23 (wild type N-Htt) ((A,B) upper panels) and pHM6-Q74 (mutant N-Htt) ((A,B) lower panels) plasmids, and the overexpression of N-Htt fragments was measured after 72 h. Representative images show cells immunolabeled with anti-HA-tag/Alexa Fluor 568 for Q23 and Q74. Hoescht 33342 was used to stain the cell nuclei. Arrows on inset images indicate toxic N-Htt aggregates. (C) Untransfected cells were used for transfection control and immunolabeled as the transfected cells. (D) Quantitative analysis was performed using the “Spot Analysis” module of the Harmony 4.9 software. The insoluble mutant N-Htt fragments were segmented on the Alexa 568 channel. The numbers of shDrp1 analyzed cells were 23351 untransfected, 49701 pHM6-Q23, and 52920 pHM6-Q74 transfected cells. The numbers of control cells analyzed were as follows: 10945 untransfected, 39873 pHM6-Q23, and 43273 pHM6-Q74 transfected cells. Data are expressed as means ± SD of n = 5 independent experiments. Groups were compared using One-way ANOVA with multiple comparisons (* indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001, and ns indicates not significant).
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Grants and funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This study was supported by the University of Debrecen, Debrecen, Hungary by a Bridging Fund (1G3D BKJ0 BFTK 247), by a fund from the International Education Office, University of Debrecen, Debrecen, Hungary for the corresponding author and by the Tempus Foundation, Stipendium Hungaricum for MG, HA, and KT.