Stage-specific control of oligodendrocyte survival and morphogenesis by TDP-43
- The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, United States
- Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg University, Germany
- Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Germany
- Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen, Germany
- Electron Microscopy Core Unit, Max-Planck-Institute of Experimental Medicine, Germany
- Kavli Neuroscience Discovery Institute, Johns Hopkins University, United States
Figures
Figure 1 with 1 supplement
Oligodendrocyte precursor cells (OPCs) require TDP-43 for survival.
(A) Schematics of CreER activation. At P90, tamoxifen was administered for five consecutive days. Tissue samples were collected 14, 30, and 60 days after the last day of tamoxifen injection. (B) Pdgf…
Figure 1—figure supplement 1
PDGFRα-TDP43 cKO shows a regional difference in degeneration of oligodendrocyte precursor cells (OPCs).
(A) Quantification of NG2+Ki-67+ proliferating OPCs in Figure 1F (unpaired, two-tailed Student’s t-test, ***p value = 0.0002, n = 3,2). (B) Coronal brain sections immunostained for GFAP at 30 and 60 …
Figure 2 with 5 supplements
Early deletion of TDP-43 in premyelinating oligodendrocytes leads to seizure development, premature death, and increased oligodendrocyte turnover.
(A) Diagram of oligodendrocyte development showing Mobp-iCre induces Tardbp deletion at the premyelinating oligodendrocyte stage whereas Mog-iCre targets myelinating oligodendrocytes. (B) …
Figure 2—figure supplement 1
Quantification of Mog-iCre recombination efficiency.
(A) Maximum intensity z-projection confocal images of the motor cortex from Mog-iCre × RCE mice at P90 and P180 immunostained with GFP and ASPA. (B) Quantification of % recombination …
Figure 2—figure supplement 2
Characterization of Mobp-iCre and Mobp-iCreERT2 mouse lines.
(A) Schematic of tamoxifen injection in different Mobp-iCreER mouse lines at P35. (B) Representative images of ASPA (oligodendrocyte marker) and NG2 (oligodendrocyte precursor cell [OPC] marker) …
Figure 2—figure supplement 3
Mobp-TDP43 and Mog-TDP43 mouse lines exhibit differences in response to loss of TDP-43.
(A) Mobp and Mog expression in oligodendrocyte lineage cells (adapted from https://greenberg.hms.harvard.edu/gene-database/ Hrvatin et al., 2018). (B) Mobp and Mog expression in the lineage of …
Figure 2—figure supplement 4
Differential gene expression between premyelinating (orange) and myelinating (blue) oligodendrocytes (OL) (reproduced from the raw single-cell RNA-Seq dataset in Hrvatin et al., 2018).
Top 16 differentially expressed genes are shown. All genes are statistically significantly differentially expressed (q value <0.01).
Figure 2—figure supplement 5
Mobp-TDP43 KO animals exhibit a higher oligodendrocyte turnover rate and increased astrogliosis.
(A) In situ hybridization for lncOL1 (9630013A20Rik) shows increased number of premyelinating oligodendrocytes in P150 Mobp-TDP43 KO compared to the Control. (B) Immunostaining for GFAP in the motor …
Figure 3 with 5 supplements
Loss of TDP-43 oligodendrocytes leads to fewer and thinner myelin sheaths.
(A) Representative transmission electron micrographs of individual axon cross-sections from Mobp-TDP43 and Mog-TDP43 mouse lines at P90 show decreased myelin sheath thickness (yellow) in the KO. …
Figure 3—figure supplement 1
Both Mobp-TDP43 KO and Mog-TDP43 KO brains show significantly increased g-ratio and aberrant myelination.
(A) Tiled mouse brain coronal sections from P90 Mobp-TDP43 and Mog-TDP43 mouse lines immunostained with myelin basic protein (MBP). Red box indicates where transmission electron microscopy (TEM) was …
Figure 3—figure supplement 2
Immunostaining for GFP, GFAP, and myelin basic protein (MBP) at P30 in Mobp-TDP43 and Mog-TDP43 mouse lines shows that oligodendrocytes and myelin are formed without astrogliosis.
Figure 3—figure supplement 3
Morphological reconstruction of individual oligodendrocytes and their myelin sheaths.
(A) Representative images of individually traced oligodendrocytes (EGFP) and myelin sheaths (myelin basic protein, MBP) from motor cortex of P180 Mog-TDP43 Control and KO. (B) Quantification of the …
Figure 3—figure supplement 4
Diagram of Control (green) and KO (red) oligodendrocytes, where KO oligodendrocytes are making fewer and thinner myelin sheaths.
Figure 3—figure supplement 5
TDP-43 loss in oligodendrocytes leads to abnormal nodal structures.
(A) Examples of normal (yellow arrowheads) and abnormal (red arrowheads) nodal and paranodal proteins (Caspr, βIV-spectrin, and myelin basic protein [MBP]) in Control and KO at P90. (B) Diagram of …
Figure 4 with 5 supplements
Oligodendrocytes without TDP-43 undergo aberrant morphological changes and exhibit aberrant wrapping of nonaxonal structures.
(A) Representative images of single oligodendrocytes from Control (Mobp-EGFP) and Mobp-TDP43 KO at P180. Scale bar = 25 μm. (B) Examples of abnormal structures formed by TDP-43 KO oligodendrocytes. …
Figure 4—figure supplement 1
Oligodendrocytes undergo progressive morphological changes with loss of TDP-43.
EGFP images from the motor cortex of Mobp-TDP43-RCE and Mog-TDP43-RCE mouse lines at P30, P90, and P180 show progressive changes in EGFP+ oligodendrocyte morphology where there is no difference at …
Figure 4—figure supplement 2
Abnormal oligodendrocyte morphological phenotype due to loss of TDP-43 is persistent throughout the CNS.
(A) Tiled coronal mouse brain section to show that hypertrophic oligodendrocyte changes can be seen throughout the brain at P180. (B) EGFP immunoreactivity shows that abnormal oligodendrocyte …
Figure 4—figure supplement 3
Longitudinal two-photon in vivo imaging of Mobp-TDP43 KO oligodendrocytes across 14 days.
Figure 4—figure supplement 4
Aberrant wrapping of non-axonal structures is prevalent in Mobp-TDP43 KO brains.
(A) Motor cortex of P180 Mobp-TDP43 KO shows that aberrant EGFP structures can be readily identified. Blue arrowheads indicate tubing-like structures that resemble blood vessel wrapping and yellow …
Figure 4—figure supplement 5
Loss of TDP-43 in oligodendrocytes potentially contributes to the disruption of blood-brain barrier (BBB) and de-differentiation of oligodendrocytes.
(A) Immunostaining for aquaporin four shows that EGFP+ wrapping displaces aquaporin 4 + astrocyte endfeet (yellow arrowheads). (B) Ki-67+NG2− oligodendrocytes can be found in P180 Mobp-TDP43 KO.
Figure 5 with 1 supplement
Adult loss of TDP-43 in oligodendrocytes leads to hindlimb weakness.
(A) Mobp-iCreER allows Tardbp deletion at mature oligodendrocytes in the adult CNS. Schematics of CreER activation where tamoxifen is administered for five consecutive days at P90. Samples are …
Figure 5—figure supplement 1
Loss of TDP-43 in oligodendrocytes does not result in degeneration of spinal motor neurons.
(A) ChAT immunostaining in the spinal cord to visualize motor neurons in the ventral spinal cord. (B) Quantification of the number of ChAT+ motor neurons per 35 μm section of the spinal cord …
Figure 6 with 5 supplements
TDP-43 maintains oligodendrocyte transcriptional integrity by suppressing cryptic exon incorporation in key oligodendrocyte genes.
(A) Bulk RNA-Seq was performed with fluorescence-activated cell sorting (FACS)-isolated EGFP+ oligodendrocytes from P30 Mobp-TDP43-RCE and Mog-TDP43-RCE cortices. (B) Volcano plots comparing KO to …
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Figure 6—source data 1
Raw, cropped, and annotated images of the western blot of mouse-Ermin (red; IRDye 680RD) and rabbit-GAPDH (green; IRDye 800CW).
The molecular weight of Ermin is 42 kDa and of GAPDH is 37 kDa.
- https://cdn.elifesciences.org/articles/75230/elife-75230-fig6-data1-v2.zip
Figure 6—figure supplement 1
The number of genes that are statistically significantly differentially expressed between different genotypes (false discovery rate FDR <0.05).
Figure 6—figure supplement 2
Gene ontology (GO) analysis from differentially expressed genes shows that Mobp-TDP43 KO and Mog-TDP43 KO show differential enrichment of biological processes.
Figure 6—figure supplement 3
Immunostaining for Ermin shows that KO oligodendrocytes have reduced cell body expression of Ermin protein compared to the Control.
Figure 6—figure supplement 4
Fragments per kilobase of exon per million mapped fragments (FPKM) of Gpr37, Mag, Gamt, and Opalin in Mobp-TDP43 Control and Mog-TDP43 Control from bulk RNA-Seq in Figure 6.
Unpaired, two-tailed Student’s t-test was used to determine statistical significance at p value <0.05.
Figure 6—figure supplement 5
Summary diagram of how TDP-43 exerts a stage-specific role in oligodendrocyte lineage cells.
Red indicates the degree of effect with the genetic loss of TDP-43. Gray bars indicate the outcomes not addressed and quantified in the conditional deletion of TDP-43 in adult oligodendrocytes.
Videos
Video 1
Video of Mobp-TDP43 Control and KO mice where Mobp-TDP43 KO mice undergo spontaneous seizures and reduced threshold for seizures.
Video 2
Longitudinal in vivo imaging of Mobp-TDP43 KO oligodendrocytes.
Video 3
3D video of EGFP+ KO oligodendrocyte that wrap around NeuN+ neuronal somata.
Video 4
Video of Mobp-TDP43 Control and cKO mice where Mobp-TDP43 cKO mice exhibit hindlimb paralysis, limp tail, and hindlimb clasping.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus, C57BL6) | Pdgfra-CreER | Kang et al., 2010 | ||
| Strain, strain background (Mus musculus, C57BL6) | Tardbpfloxed | Chiang et al., 2010 | ||
| Strain, strain background (Mus musculus, Mixed) | RCE (Rosa-CAG-LSL-EGFP) | Sousa et al., 2009 | JAX MMRRC stock #32,038 | |
| Strain, strain background (Mus musculus, C57BL6) | Mobp-iCre | This paper | Generated and maintained by D.E. Bergles lab | |
| Strain, strain background (Mus musculus, C57BL6) | Mobp-iCreER | This paper | Generated and maintained by D.E. Bergles lab | |
| Strain, strain background (Mus musculus, C57BL6) | MogiCre | Buch et al., 2005 | ||
| Strain, strain background (Mus musculus, C57BL6) | STOCK Tg (Mobp-EGFP) IN1Gsat/Mmucd | MMRC | RRID:MMRRC_030483- UCD | Hughes et al., 2018 |
| Antibody | Anti-Aspartoacylase (ASPA) (rabbit polyclonal) | Gentex | RRID:AB_2036283 | (1:1500) |
| Antibody | Anti-GFP (chicken polyclonal) | Aves lab | RRID:AB_2307313 | (1:1500) |
| Antibody | Anti-MBP (mouse monoclonal) | Sternberger | RRID:AB_2564741 | (1:2000) |
| Antibody | Anti-NG2 (guinea pig serum) | Generated in D.E. Bergles lab against entire NG2 protein Kang et al., 2013 | (1:10,000) | |
| Antibody | Anti-Ki-67 (rabbit polyclonal) | Abcam | RRID:AB_443209 | (1:1000) |
| Antibody | Anti-GFP (goat polyclonal) | Sicgen | RRID:AB_2333099 | (1:7500) |
| Antibody | Anti-MBP (chicken polyclonal) | Aves lab | RRID:AB_2313550 | (1:500) |
| Antibody | Anti-GFAP (rabbit polyclonal) | Agilent | RRID:AB_10013482 | (1:500) |
| Other | Alexa Fluor 647 Isolectin GS-IB4 | Thermo Fisher | (1:200) | |
| Antibody | Anti-Aquaporin 4 (rabbit polyclonal) | Sigma | RRID:AB_1844967 | (1:1500) |
| Antibody | Anti-NeuN (mouse monoclonal) | Millipore | RRID:AB_2298772 | (1:500) |
| Antibody | Anti-Ermin (mouse monoclonal) | Generated by E. Peles Lab | Gift from E. Peles | (1:1000) |
| Antibody | Anti-Caspr (guinea pig polyclonal) | Generated by M. Bhat Lab | Gift from M. Bhat | (1:1500) |
| Antibody | Anti-βIV spectrin (rabbit polyclonal) | Generated by M. Rasband Lab | Gift from M. Rasband | (1:300) |
| Antibody | Anti-ChAT (goat polyclonal) | Millipore | RRID:AB_90650 | (1:500) |
| Antibody | Anti-CC1 (mouse monoclonal) | Millipore | RRID:AB_2242783 | (1:50) |
| Antibody | Anti-GAPDH (rabbit monoclonal) | Cell Signalling | RRID:AB_10622025 | (1:1000) |
| Antibody | Anti-Rabbit IgG conjugated to Cy3 or Cy5 (donkey polyclonal) | Jackson Immuno | RRID:AB_2313568RRID:AB_2340625 | (1:2000) |
| Antibody | Anti-Mouse IgG conjugated to Cy3 or Cy5 (donkey polyclonal) | Jackson Immuno | RRID:AB_2340817RRID:AB_2340820 | (1:2000) |
| Antibody | Anti-Guinea pig IgG conjugated to Cy3 or Cy5 (donkey polyclonal) | Jackson Immuno | RRID:AB_2340461RRID:AB_2340477 | (1:2000) |
| Antibody | Anti-Goat IgG conjugated to Alexa 488 or Cy3 (donkey polyclonal) | Jackson Immuno | RRID:AB_2340430RRID:AB_2340413 | (1:2000) |
| Antibody | Anti-Chicken IgG conjugated to Alexa 488 or Cy5 (donkey polyclonal) | Jackson Immuno | RRID:AB_2340376RRID:AB_2340347 | (1:2000) |
| Software, algorithm | ZEN Blue/Black | Zeiss | RRID:SCR_013672 | |
| Software, algorithm | Fiji | http://fiji.sc | RRID:SCR_002285 | |
| Chemical compound, drug | Tamoxifen | Sigma | ||
| Chemical compound, drug | Sunflower seed oil | Sigma |
Additional files
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Supplementary file 1
Differential gene expression of TDP-43 KO oligodendrocytes.
Excel file of bulk RNA-Seq that includes average FPKM of each gene, log fold change (FC), p value, and false discovery rate (FDR) for the following comparisons: (1) Mog-TDP43-WT vs. Mobp-TDP43-WT, (2) Mog-TDP43-KO vs. Mobp-TDP43-KO, (3). Mog-TDP43-KO vs. Mog-TDP43-WT, (4) Mobp-TDP43-KO vs. Mobp-TDP43-WT, and (5) TDP43-WT vs. TDP43-KO.
- https://cdn.elifesciences.org/articles/75230/elife-75230-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/75230/elife-75230-transrepform1-v2.docx
