Promoting axon regeneration by inhibiting RNA N6-methyladenosine demethylase ALKBH5
- Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, NMPA Key Laboratory for Research and Evaluation of Tissue Engineering Technology Products, Co-innovation Center of Neuroregeneration, Nantong University, China
- Jiangsu Key Laboratory of Neuropsychiatric Diseases and Institute of Neuroscience, Soochow University; Clinical Research Center of Neurological Disease, The Second Affiliated Hospital of Soochow University, China
Figures
Figure 1 with 1 supplement
Reduced ALKBH5 in dorsal root ganglions (DRGs) enhances neurite outgrowth.
(A) Diagrammatic representation of for dynamic and reversible RNA m6A modification. The m6A modification can be added by ‘writers’ (METTL3/14, WTAP complex), demethylated by ‘erasers’ (FTO and …
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Figure 1—source data 1
The data underlying all the graphs shown in Figure 1.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig1-data1-v1.xlsx
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Figure 1—source data 2
The data underlying all the graphs shown in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig1-data2-v1.xlsx
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Figure 1—source data 3
Source files for ALKBH5 western graphs.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig1-data3-v1.zip
Figure 1—figure supplement 1
Validation of the interfering efficiency for the indicated m6A-related gene and the distribution of the ALKBH5 in different dorsal root ganglion (DRG) neurons.
(A) Experimental setup. Mature DRG neurons were transfected with the control or target siRNA for 2 d, and axon staining was conducted 16–18 hr after cell replating. (B) Examination of the siRNA …
Figure 2 with 1 supplement
ALKBH5 inhibits axonal regeneration in an m6A-dependent manner.
(A) ALKBH5 protein expression by western blot. Protein extracts isolated from dissociated adult dorsal root ganglion (DRG) neurons infected with the EGFP (control), wild type ALKBH5 (wt-ALKBH5), and …
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Figure 2—source data 1
The data underlying all the graphs shown in Figure 2.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig2-data1-v1.xlsx
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Figure 2—source data 2
The data underlying all the graphs shown in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig2-data2-v1.xlsx
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Figure 2—source data 3
Source files for ALKBH5 western graphs.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig2-data3-v1.zip
Figure 2—figure supplement 1
Infection efficiency of AAV2/8 in dorsal root ganglion (DRG) by intrathecal injection.
(A) DRG sections from rat after intrathecal injection of control, wt-ALKBH5, and mut-ALKBH5 adeno-associated viruses (AAVs) for 2 wk. The sections were stained for EGFP (green) and Tuj1 (red). Scale …
Figure 3 with 3 supplements
ALKBH5 deficiency promotes axon regeneration.
(A) ALKBH5 protein expression by western blot. Protein extracts isolated from dissociated adult dorsal root ganglion (DRG) neurons transfected with the shControl (NC), shAlkbh5-1 (KD1), and shAlkbh5-…
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Figure 3—source data 1
The data underlying all the graphs shown in Figure 3.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig3-data1-v1.xlsx
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Figure 3—source data 2
The data underlying all the graphs shown in Figure 3—figure supplements 1–3.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig3-data2-v1.xlsx
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Figure 3—source data 3
Source files for ALKBH5 western graphs.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig3-data3-v1.zip
Figure 3—figure supplement 1
ALKBH5 knockdown promotes dorsal root ganglion (DRG) neurite outgrowth in the present of chondroitin sulfate proteoglycan (CSPG).
(A) Representative images of replated DRG neurons from NC, KD1, and KD2 groups incubated with CSPG substrates with Tuj1 staining. DRG neurons were dissociated and infected with the NC, KD1, and KD2 …
Figure 3—figure supplement 2
Infection efficiency of AAV2/8 in dorsal root ganglion (DRG) by intrathecal injection.
(A) DRG sections from rat after NC, KD1, and KD2 adeno-associated viruses (AAVs) intrathecal injection for 2 wk. The sections were stained for EGFP (green) and Tuj1 (red). Scale bar: 100 µm. (B) …
Figure 3—figure supplement 3
The selective ALKBH5 inhibitors (SAI) promoted axon regeneration of dorsal root ganglion (DRG) neurons.
(A, B) CCK-8 assay showing the viability of cultured DRG neurons treated with various concentrations of selective ALKBH5 inhibitors (Z56957173 or Z52453295) for 24 hr (One -way ANOVA followed by …
Figure 4 with 1 supplement
Lpin2 is a target of ALKBH5-mediated axonal regeneration.
(A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses for differential gene expression of dorsal root ganglion (DRG) neurons infected with NC or KD2. The total RNA extracts isolated …
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Figure 4—source data 1
The data underlying all the graphs shown in Figure 4.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig4-data1-v1.xlsx
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Figure 4—source data 2
The data underlying all the graphs shown in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig4-data2-v1.xlsx
Figure 4—figure supplement 1
Validation of the interference efficiency for ALKBH5-induced differential gene expression.
Quantification of mRNA expression by qRT-PCR validating the knockdown efficiency of the ALKBH5-induced differentially expressed genes (Aldh3b1, Lpin2, and Galns) in dorsal root ganglion (DRG) …
Figure 5 with 3 supplements
ALKBH5 impacts Lpin2 mRNA stability through its RNA demethylase activity.
(A, B) Changes in the m6A-modified Lpin2 level with ALKBH5 knockdown or overexpression. RNA extracts isolated from dissociated adult dorsal root ganglion (DRG) neurons infected with NC, KD1, and KD2 …
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Figure 5—source data 1
The data underlying all the graphs shown in Figure 5.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig5-data1-v1.xlsx
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Figure 5—source data 2
The data underlying all the graphs shown in Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig5-data2-v1.xlsx
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Figure 5—source data 3
The data underlying all the graphs shown in Figure 5—figure supplement 2.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig5-data3-v1.xlsx
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Figure 5—source data 4
Source files for LPIN2 and ALKBH5 western graphs.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig5-data4-v1.zip
Figure 5—figure supplement 1
ALKBH5 has no impact on Lpin2 pre-mRNA stability during the axonal regeneration.
(A) Enrichment of Lpin2-Pre mRNA m6A-level in dorsal root ganglion (DRG) neurons at days 0 and day 3 following sciatic nerve crush (SNC). Total RNA extracts isolated from dissociated adult DRG …
Figure 5—figure supplement 2
ALKBH5 did not affected the nuclear export of Lpin2 or Lpin2-pre mRNA during the axonal regeneration.
(A–D) The distributed proportion of Lpin2 mRNA or Lpin2-Pre mRNA in dorsal root ganglion (DRG) neurons nucleus and cytoplasm RNA isolated from dissociated adult DRG neurons transfected with NC, KD1, …
Figure 5—figure supplement 3
The m6A modification sites on Lpin2 mRNA predicted by SRAMP.
Blue frame represents the 3′ UTR region of Lpin2, and red circle represents the m6A motif GGACA located near the stop codon in the 3′ UTR.
Figure 6 with 1 supplement
LPIN2 impairs axon regeneration.
(A) Quantification of Lpin2 mRNA expression by qRT-PCR in adult rat L4-5 DRGs following sciatic nerve crush (SNC). Gapdh was used as the internal control. Adult dorsal root ganglions (DRGs) were …
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Figure 6—source data 1
The data underlying all the graphs shown in Figure 6.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig6-data1-v1.xlsx
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Figure 6—source data 2
The data underlying all the graphs shown in Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig6-data2-v1.xlsx
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Figure 6—source data 3
Source files for LPIN2 western graphs.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig6-data3-v1.zip
Figure 6—figure supplement 1
Infection efficiency of AAV2/8 in dorsal root ganglion (DRG) by intrathecal injection.
(A) DRG sections from rat after intrathecal injection of control and LPIN2 adeno-associated viruses (AAVs) for 2 wk. The sections were stained for EGFP (green) and Tuj1 (red). Scale bar: 100 µm. (B) …
Figure 7
LPIN2 reverses ALKBH5 deficiency-induced axonal regeneration following sciatic nerve crush (SNC).
(A) Representative images of replated dorsal root ganglion (DRG) neurons from NC + EGFP, KD2 + EGFP, and KD2 + LPIN2 groups with Tuj1 staining. DRG neurons were dissociated and infected with the NC …
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Figure 7—source data 1
The data underlying all the graphs shown in Figure 7.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig7-data1-v1.xlsx
Figure 8 with 1 supplement
ALKBH5 inhibition promotes retinal ganglion cell (RGC) survival and optic nerve regeneration after optic nerve crush (ONC).
(A) Retinal sections from adult mice from day 0 (Intact) and day 3 (3d) following ONC were collected and stained with ALKBH5 and Tuj1. Red for Tuj1, green for ALKBH5; scale bar: 50 μm. (B) Relative …
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Figure 8—source data 1
The data underlying all the graphs shown in Figure 8.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig8-data1-v1.xlsx
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Figure 8—source data 2
The data underlying all the graphs shown in Figure 8—figure supplement 1.
- https://cdn.elifesciences.org/articles/85309/elife-85309-fig8-data2-v1.xlsx
Figure 8—figure supplement 1
Infection efficiency of the AAV2/2 in retinal ganglion cells (RGCs) by intravitreal injection.
(A) Retinal sections from mice after AAV2/2 intravitreal injection for 2 wk. Retinal sections were stained for EGFP (green) and Tuj1 (red); scale bar: 50 µm. (B) Percentage of EGFP-positive RGCs …
Author response image 1
The prediction result for miR-221 binding with the 3'UTR region of Alkbh5.
Author response image 2
YTHDF3 knockdown did not impact the Lpin2 mRNA and protein expression.
(A) Quantification of Lpin2 mRNA expression by qRT-PCR analysis of total RNA extracts isolated from dissociated adult DRG neurons infected with the shControl, shYthdf3-1, or shYthdf3-2 AAVs for 7 …
Author response image 3
Kidney sections from adult rat stained with ALKBH5 antibody (Abcam, Cat# ab195377) (green) and DAPI (blue); scale bar: 25 μm.
Author response image 4
The viability of cultured DRG neurons infected with various AAVs.
(A, and B) DRG neurons were infected with various AAVs (Control, wt-ALKBH5, mut-ALKBH5 or NC, KD1, KD2) for 16 hours, cultured for 7 days and replated for another 16 hours. Then the cell viability …
Author response image 5
The viability of cultured DRG neurons infected with AAVs overexpressing LPIN2.
DRG neurons were infected with EGFP or LPIN2 AAVs for 16 hours, cultured for 7 days and replated for another 16 hours. Then the cell viability was examined by CCK-8 assay. Unpaired two-tailed …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Rattus norvegicus) | Alkbh5 | GenBank | Gene ID: 303193 | |
| Gene (R. norvegicus) | Lpin2 | GenBank | Gene ID: 316737 | |
| Cell line (Homo sapiens) | HEK-293T | ATCC | CRL-3216 | |
| Strain, strain background (AAV) | AAV2/8-CMV bGlobin-eGFP-U6-shRNA | OBiO | Express control shRNA in DRG | |
| Strain, strain background (AAV) | AAV2/8-CMV bGlobin-eGFP-U6-Alkbh5 shRNA1 | OBiO | Express Alkbh5 shRNA-1 in DRG | |
| Strain, strain background (AAV) | AAV2/8-CMV bGlobin-eGFP-U6-Alkbh5 shRNA2 | OBiO | Express Alkbh5 shRNA-2 in DRG | |
| Strain, strain background (AAV) | AAV2/8-CMV-3xFLAG-P2A-mNeonGreen-tWPA | OBiO | Express EGFP in DRG | |
| Strain, strain background (AAV) | AAV2/8-CMV-LPIN2-3xFLAG-P2A-mNeonGreen-tWPA | OBiO | Express LPIN2 in DRG | |
| Strain, strain background (AAV) | AAV2/8-hSyn-EGFP-WPRE-hGH polyA | BrianVTA | Express EGFP in DRG | |
| Strain, strain background (AAV) | AAV2/8-hSyn-ALKBH5-EGFP-WPRE-hGH polyA | BrianVTA | Express wild type rat ALKBH5 in DRG | |
| Strain, strain background (AAV) | AAV2/8-hSyn-ALKBH5(H205D)-EGFP-WPRE-hGH polyA | BrianVTA | Express mutant rat ALKBH5 in DRG | |
| Strain, strain background (AAV) | AAV2/2-CMV bGlobin-eGFP-U6-shRNA | OBiO | Express control shRNA in RGC | |
| Strain, strain background (AAV) | AAV2/2-CMV bGlobin-eGFP-U6-Alkbh5 shRNA | OBiO | Express Alkbh5 shRNA in RGC | |
| Antibody | Anti-ALKBH5 (rabbit monoclonal) | Abcam | Cat# ab195377; RRID:AB_2827986 | IF (1:500), WB (1:1000) |
| Antibody | Anti-Tuj1 (mouse monoclonal) | R&D Systems | Cat# MAB1195; RRID:AB_357520 | IF (1:500) |
| Antibody | Anti-LPIN2 (rabbit monoclonal) | Abcam | Cat# ab176347; RRID:AB_2924332 | WB (1:1000) |
| Antibody | Anti-SCG10 (rabbit polyclonal) | Novus | Cat# NBP1-49461; RRID:AB_10011569 | IF (1:500) |
| Antibody | Anti-m6A (rabbit polyclonal) | Synaptic Systems | Cat# 202003; RRID:AB_2279214 | MeRIP (1:100) |
| Antibody | Anti-IgG (rabbit monoclonal) | Abcam | Cat# ab172730; RRID:AB_2687931 | MeRIP (1:100) |
| Antibody | Anti-GFP (chicken polyclonal) | Abcam | Cat# ab13970; RRID:AB_300798 | IF (1:500) |
| Antibody | Anti-GAPDH (rabbit polyclonal) | Sigma | Cat# G9545; RRID:AB_796208 | WB (1:5000) |
| Antibody | Anti-mouse-647 secondary antibody (goat polyclonal) | Invitrogen | Cat# A32728; RRID:AB_2633277 | IF (1:1000) |
| Antibody | Anti-rabbit-488 secondary antibody (donkey polyclonal) | Invitrogen | Cat# A21206; RRID:AB_2535792 | IF (1:1000) |
| Antibody | Anti-rabbit IgG H&L(goat polyclonal) | Jackson ImmunoResearch Labs | Cat# 111-035-003; RRID:AB_2313567 | WB (1:5000) |
| Antibody | Anti-chicken-488 secondary antibody (goat polyclonal) | Abcam | Cat# ab150169; RRID:AB_2636803 | IF (1:1000) |
| Antibody | Anti-rabbit-594 secondary antibody (goat polyclonal) | Invitrogen | Cat# A11012; RRID:AB_2534079 | IF (1:1000) |
| Peptide, recombinant protein | Cholera Toxin Subunit B (Recombinant), Alexa Fluor 555 Conjugate | Invitrogen | Cat# C34776 | 1 μg/μL |
| Recombinant DNA reagent | pmirGLO–Lpin2(rat)–3′ UTR-wt (plasmid) | Miaoling Biology | P25026 | |
| Recombinant DNA reagent | pmirGLO–Lpin2(rat)–3′ UTR-mut (plasmid) | Miaoling Biology | P25121 | |
| Commercial assay or kit | SELECT m6A site identification kit | Epibiotek | R202106M-01-10T | Detect the m6A level of m6A motif (GGACA) in Lpin2-3’ UTR region |
| Chemical compound, drug | Z52453295 | Enamine | CAS:956935-02-7 | 0, 5, 10, 20, 40, 80 μM (in vitro); 0, 1.56, 6.25, 25, 100 mM (in vivo) |
| Chemical compound, drug | Z56957173 | Enamine | CAS:37510-29-5 | 0, 0.5, 1, 2, 5 μM (in vitro) |
| Software, algorithm | ImageJ software | NIH | https://imagej.nih.gov/ij/ | |
| Software, algorithm | GraphPad Prism 8 | GraphPad Software | https://www.graphpad.com |
Additional files
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Supplementary file 1
Primers for qRT-PCR validation of candidate Genes.
- https://cdn.elifesciences.org/articles/85309/elife-85309-supp1-v1.docx
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Supplementary file 2
Interference sequence (IS) designed for target gene.
- https://cdn.elifesciences.org/articles/85309/elife-85309-supp2-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/85309/elife-85309-mdarchecklist1-v1.docx
