Stage-specific control of oligodendrocyte survival and morphogenesis by TDP-43
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Evaluation Summary:
The manuscript provides strong support for the concept that oligodendrocyte alterations contribute to neurological disorders that were previous thought to be primarily cell autonomous to neurons. The work is very well done, the results presented are clear and convincing, and the discussion is reasonable and interesting. The study will have considerable impact on the assessment of various neurodegenerative disorders with TDP-43 alterations.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Generation of oligodendrocytes in the adult brain enables both adaptive changes in neural circuits and regeneration of myelin sheaths destroyed by injury, disease, and normal aging. This transformation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes requires processing of distinct mRNAs at different stages of cell maturation. Although mislocalization and aggregation of the RNA-binding protein, TDP-43, occur in both neurons and glia in neurodegenerative diseases, the consequences of TDP-43 loss within different stages of the oligodendrocyte lineage are not well understood. By performing stage-specific genetic inactivation of Tardbp in vivo, we show that oligodendrocyte lineage cells are differentially sensitive to loss of TDP-43. While OPCs depend on TDP-43 for survival, with conditional deletion resulting in cascading cell loss followed by rapid regeneration to restore their density, oligodendrocytes become less sensitive to TDP-43 depletion as they mature. Deletion of TDP-43 early in the maturation process led to eventual oligodendrocyte degeneration, seizures, and premature lethality, while oligodendrocytes that experienced late deletion survived and mice exhibited a normal lifespan. At both stages, TDP-43-deficient oligodendrocytes formed fewer and thinner myelin sheaths and extended new processes that inappropriately wrapped neuronal somata and blood vessels. Transcriptional analysis revealed that in the absence of TDP-43, key proteins involved in oligodendrocyte maturation and myelination were misspliced, leading to aberrant incorporation of cryptic exons. Inducible deletion of TDP-43 from oligodendrocytes in the adult central nervous system (CNS) induced the same progressive morphological changes and mice acquired profound hindlimb weakness, suggesting that loss of TDP-43 function in oligodendrocytes may contribute to neuronal dysfunction in neurodegenerative disease.
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Author Response:
Reviewer #3 (Public Review):
In both neurons and glia (astrocytes, microglia, and oligodendrocytes) of patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD), the DNA/RNA-binding protein TDP-43 is mislocalized from the nucleus to the cytoplasm where it forms pathological inclusions. Because this subcellular redistribution leads to TDP-43 depletion from the nucleus, the pathogenic mechanism may involve (1) the loss of nuclear function, (2) a gain of cytoplasmic function, or (3) a contribution from both. Heo, Dongeun et al., teases apart this first possibility by investigating the depletion of TDP-43 within specific stages of the oligodendrocyte lineage in vivo and raising the possibility of glial damage in disease progression. The authors found that the consequences of TDP-43 deletion …
Author Response:
Reviewer #3 (Public Review):
In both neurons and glia (astrocytes, microglia, and oligodendrocytes) of patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD), the DNA/RNA-binding protein TDP-43 is mislocalized from the nucleus to the cytoplasm where it forms pathological inclusions. Because this subcellular redistribution leads to TDP-43 depletion from the nucleus, the pathogenic mechanism may involve (1) the loss of nuclear function, (2) a gain of cytoplasmic function, or (3) a contribution from both. Heo, Dongeun et al., teases apart this first possibility by investigating the depletion of TDP-43 within specific stages of the oligodendrocyte lineage in vivo and raising the possibility of glial damage in disease progression. The authors found that the consequences of TDP-43 deletion in oligodendrocytes was dependent on the stage of oligodendrocyte maturation. First, they find that deletion of TDP-43 from oligodendrocyte precursor cells (OPCs) resulted in their rapid death, however OPCs that retained TDP-43 expression repopulated to their normal density. Secondly, they find that constitutive deletion of TDP-43 from early premyelinating oligodendrocytes exhibited seizures and early lethality. Similar conditional deletion of TDP-43 from early premyelinating oligodendrocytes in the adult CNS induces abnormal morphological changes, motor discoordination (but no seizures), and premature lethality. Meanwhile, constitutive deletion of TDP-43 from myelinating oligodendrocytes did not lead to any gross phenotypes or shortened lifespan. However, early deletion led to oligodendrocyte degeneration and astrogliosis followed by oligodendrocyte regeneration. Interestingly, in both early and mature oligodendrocytes loss of TDP-43 lead to morphological changes, thinner myelin, less myelinated axons, and aberrant myelination. These results are very interesting and opens the door for further questions, such as why are the oligodendrocytes mislocalizing their myelination targets? Why does early deletion of TDP-43 cause such drastic phenotypes? Lastly, to understand the molecular consequences of TDP-43 deletion in both early and late myelinating oligodendrocytes the authors perform RNAseq on FACS isolated KO cells and controls. The authors uncover that loss of TDP-43 from oligodendrocytes in the adult CNS leads to altered splicing of key regulators of oligodendrocyte growth and morphogenesis.
These findings complement those of Wang, J et al 2018 that shows depletion of TDP-43 in mature oligodendrocytes in the spinal cord is indispensable for the proper functioning of mature oligodendrocytes, including myelination and cell survival. Although Wang, J et al 2018 saw no apparent harm to motor neurons of mice with TDP-43 deleted in mature oligodendrocytes, the mice did have progressive motor deficits and early lethality - similar to Heo, Dongeun et al.
Strengths:
To investigate the role of TDP-43 within distinct stages of oligodendrocyte maturation, the authors used four different Cre and CreER mouse lines: 1) Pdgfra-CreERT2, 2) Mobp-iCre, 3) Mog-iCre, and 4) Mobp-iCreERT2, thus allowing them to inactive Tardbp in both the developing and mature CNS. By performing thorough analysis at each discrete stage within the oligodendrocyte lineage, the authors uncovered differential requirement for TDP-43 in cell survival and structural maintenance as OPCs transform into early and late myelinating oligodendrocytes. These results are important because they elucidate the contribution of a DNA/RNA-binding protein to oligodendrocyte development. Additionally, the results of the conditional deletion of TDP-43 from early premyelinating oligodendrocytes in the adult CNS is critical for understanding how nuclear depletion of TDP-43 from oligodendrocyte might contribute to disease pathogenesis.
The authors also performed bulk RNAseq on early and late myelinating oligodendrocyte controls and TDP-43 KO cells. In doing so, not only did they uncover hundreds of differentially expressed (DE) genes between each control and KO, but thousands of DE genes between the two controls. This experiment also confirmed that Mobp-iCre and Mog-iCre mouse lines were able to target different stages of oligodendrocyte development. This dataset is very exciting to both the developmental glial biology community and to those trying to understand the molecular mechanisms within glia that contribute to neurodegenerative disorders.
Minor weaknesses:
In Figure 1, the authors observe that in the cKO mice, the OPCs are dying because they observe a lack of NG2 staining. Is it possible the OPCs have changed to another cell identity that is NG2- in the absence of TDP-43? Tunnel staining would clarify that indeed the cKO OPCs are dying. Furthermore, the authors note that despite the extensive death of OPCs, they do not see signs of GFAP+ astrogliosis. Is there instead an increase in microglia activation? Throughout the paper, the authors use only GFAP+ astrogliosis to measure widespread inflammation. It would be more compelling to also look at the contribution of microglia or other inflammatory markers to measure inflammation.
To perform fate-mapping of TDP-43 deficient OPCs, we crossed the Cre-dependent EGFP reporter (RCE) to Pdgfra-CreER x Tardbp floxed mice (PDGFRα-TDP43). By assessing EGFP expression, we determined that disrupting Tardbp expression in OPCs results in rapid degeneration of OPCs within one month. The dramatic reduction in EGFP^+ OPCs indicates that knockout of Tardbp results in cell loss rather than downregulation of NG2 and/or a shift in cell identity.
We agree that it would also be informative to extend these studies to examine other markers of neuroinflammation. We settled on GFAP immunostaining for visualization purposes, as cortical GFAP immunoreactivity is limited in control mice, providing the greatest sensitivity and has been established as a robust biomarker of neuroinflammation. As the external consequences are not the primary focus of this study and are likely to be both complex and time dependent, we feel that these studies are outside the scope of the present study.
As shown in Figure 3, loss of TDP-43 in oligodendrocytes at early and mature stages leads to similar profound phenotypes within both the Mobp-TDP43KO and Mogp-TDP43KO mouse lines. However, only early when TDP-43 is deleted using the Mobp-TDP43KO, are there severe physical phenotypes in the mice and early lethality. However, the authors show that there is no change in the density of ASPA+ mature oligodendrocytes in Mobp-TDP43KO and Mogp-TDP43KO at any stage. If there is an increase in the turnover of oligodendrocytes and oligodendrocyte number stays the same, can the authors speculate in their discussion what they believe is causing the severe seizure and lethality phenotypes in the Mobp-TDP43 KO mice? The authors mention that there is an increase in astrogliosis. Are they suggesting this change in astrocyte activity could promote the severe phenotypes and early lethality? Because motor neuron number is not affected by TDP-43 deletion, but no direct measurements of motor neuron activity were taken, it is hard to make sense of the phenotypes observed.
In the Discussion, we speculated that early deletion of TDP-43 in oligodendrocytes (Mobp-TDP43 KO) compromises their long-term survival. This gradual degeneration of oligodendrocytes, which is masked at the population level by OPC mediated regeneration, nevertheless induces widespread astrogliosis, indicative of progressive neuroinflammation. Oligodendrocyte loss has previously been shown to induce neuroinflammation, seizures and neurodegeneration (Traka et al. 2016). We agree that there are many interesting features that remain to be understood about the phenotypic consequences of TDP-43 loss within oligodendrocytes, such as possible reorganization of myelin patterns (see Orthmann-Murphy, Call, et al. 2020) and mechanistic links between oligodendrocyte degeneration, gliosis and inflammation. We are pursuing some of this analysis now, but they necessitate extensive interrogation of the transgenic mice using new assays. Thus we feel that these experiments are outside the scope of the current study.
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Evaluation Summary:
The manuscript provides strong support for the concept that oligodendrocyte alterations contribute to neurological disorders that were previous thought to be primarily cell autonomous to neurons. The work is very well done, the results presented are clear and convincing, and the discussion is reasonable and interesting. The study will have considerable impact on the assessment of various neurodegenerative disorders with TDP-43 alterations.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Heo et al., 2021 determined the consequences of deleting DNA/RNA-binding protein TDP-43 on oligodendrocyte maturation and function using transgenic mouse models. While TDP-43 deletion in early oligodendrocyte maturation resulted in abnormal morphological changes and premature cell death, no changes were observed following TDP-43 deletion in mature oligodendrocytes. Transcriptomic analysis of oligodendrocytes following TDP-43 deletion revealed missplicing of cytoskeletal-associated genes, which suggests that TDP-43 plays a crucial role in oligodendrocyte cytoskeletal organization. These findings revealed TDP-43 as a key regulator of oligodendrocyte maturation, which may go awry in neurodegenerative disorders.
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Reviewer #2 (Public Review):
The investigators examined the in vivo effect of the genetic inactivation of the mouse gene that encodes TDP-43 on distinct stages of oligodendrocyte lineage cells. The authors combined a mouse strain containing a conditional allele of the Tardbp gene, which encodes the TDP-43 protein, with several Cre driver lines that induce recombination at discrete stages of oligodendrocyte development. Oligodendrocyte progenitor cells (OPCs), pre-myelinating oligodendrocytes, myelinating oligodendrocytes, as well as mature oligodendrocytes maintaining a myelin sheath were examined in these studies. Interestingly, the loss of TDP-43 results in distinct outcomes depending on the developmental stage of the oligodendrocytes at the time of genetic inactivation. The work is exceedingly well done, the results presented are …
Reviewer #2 (Public Review):
The investigators examined the in vivo effect of the genetic inactivation of the mouse gene that encodes TDP-43 on distinct stages of oligodendrocyte lineage cells. The authors combined a mouse strain containing a conditional allele of the Tardbp gene, which encodes the TDP-43 protein, with several Cre driver lines that induce recombination at discrete stages of oligodendrocyte development. Oligodendrocyte progenitor cells (OPCs), pre-myelinating oligodendrocytes, myelinating oligodendrocytes, as well as mature oligodendrocytes maintaining a myelin sheath were examined in these studies. Interestingly, the loss of TDP-43 results in distinct outcomes depending on the developmental stage of the oligodendrocytes at the time of genetic inactivation. The work is exceedingly well done, the results presented are clear and convincing and the discussion of the work is reasonable and interesting. The study will have considerable impact on the assessment of various neurodegenerative disorders with TDP-43 alterations. The work provides strong support for the concept that oligodendrocyte alterations contribute to neurological disorders that were previous thought to be primarily cell autonomous to neurons.
-
Reviewer #3 (Public Review):
In both neurons and glia (astrocytes, microglia, and oligodendrocytes) of patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD), the DNA/RNA-binding protein TDP-43 is mislocalized from the nucleus to the cytoplasm where it forms pathological inclusions. Because this subcellular redistribution leads to TDP-43 depletion from the nucleus, the pathogenic mechanism may involve (1) the loss of nuclear function, (2) a gain of cytoplasmic function, or (3) a contribution from both. Heo, Dongeun et al., teases apart this first possibility by investigating the depletion of TDP-43 within specific stages of the oligodendrocyte lineage in vivo and raising the possibility of glial damage in disease progression. The authors found that the consequences of TDP-43 deletion in oligodendrocytes …
Reviewer #3 (Public Review):
In both neurons and glia (astrocytes, microglia, and oligodendrocytes) of patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD), the DNA/RNA-binding protein TDP-43 is mislocalized from the nucleus to the cytoplasm where it forms pathological inclusions. Because this subcellular redistribution leads to TDP-43 depletion from the nucleus, the pathogenic mechanism may involve (1) the loss of nuclear function, (2) a gain of cytoplasmic function, or (3) a contribution from both. Heo, Dongeun et al., teases apart this first possibility by investigating the depletion of TDP-43 within specific stages of the oligodendrocyte lineage in vivo and raising the possibility of glial damage in disease progression. The authors found that the consequences of TDP-43 deletion in oligodendrocytes was dependent on the stage of oligodendrocyte maturation. First, they find that deletion of TDP-43 from oligodendrocyte precursor cells (OPCs) resulted in their rapid death, however OPCs that retained TDP-43 expression repopulated to their normal density. Secondly, they find that constitutive deletion of TDP-43 from early premyelinating oligodendrocytes exhibited seizures and early lethality. Similar conditional deletion of TDP-43 from early premyelinating oligodendrocytes in the adult CNS induces abnormal morphological changes, motor discoordination (but no seizures), and premature lethality. Meanwhile, constitutive deletion of TDP-43 from myelinating oligodendrocytes did not lead to any gross phenotypes or shortened lifespan. However, early deletion led to oligodendrocyte degeneration and astrogliosis followed by oligodendrocyte regeneration. Interestingly, in both early and mature oligodendrocytes loss of TDP-43 lead to morphological changes, thinner myelin, less myelinated axons, and aberrant myelination. These results are very interesting and opens the door for further questions, such as why are the oligodendrocytes mislocalizing their myelination targets? Why does early deletion of TDP-43 cause such drastic phenotypes? Lastly, to understand the molecular consequences of TDP-43 deletion in both early and late myelinating oligodendrocytes the authors perform RNAseq on FACS isolated KO cells and controls. The authors uncover that loss of TDP-43 from oligodendrocytes in the adult CNS leads to altered splicing of key regulators of oligodendrocyte growth and morphogenesis.
These findings complement those of Wang, J et al 2018 that shows depletion of TDP-43 in mature oligodendrocytes in the spinal cord is indispensable for the proper functioning of mature oligodendrocytes, including myelination and cell survival. Although Wang, J et al 2018 saw no apparent harm to motor neurons of mice with TDP-43 deleted in mature oligodendrocytes, the mice did have progressive motor deficits and early lethality - similar to Heo, Dongeun et al.
Strengths:
To investigate the role of TDP-43 within distinct stages of oligodendrocyte maturation, the authors used four different Cre and CreER mouse lines: 1) Pdgfra-CreERT2, 2) Mobp-iCre, 3) Mog-iCre, and 4) Mobp-iCreERT2, thus allowing them to inactive Tardbp in both the developing and mature CNS. By performing thorough analysis at each discrete stage within the oligodendrocyte lineage, the authors uncovered differential requirement for TDP-43 in cell survival and structural maintenance as OPCs transform into early and late myelinating oligodendrocytes. These results are important because they elucidate the contribution of a DNA/RNA-binding protein to oligodendrocyte development. Additionally, the results of the conditional deletion of TDP-43 from early premyelinating oligodendrocytes in the adult CNS is critical for understanding how nuclear depletion of TDP-43 from oligodendrocyte might contribute to disease pathogenesis.
The authors also performed bulk RNAseq on early and late myelinating oligodendrocyte controls and TDP-43 KO cells. In doing so, not only did they uncover hundreds of differentially expressed (DE) genes between each control and KO, but thousands of DE genes between the two controls. This experiment also confirmed that Mobp-iCre and Mog-iCre mouse lines were able to target different stages of oligodendrocyte development. This dataset is very exciting to both the developmental glial biology community and to those trying to understand the molecular mechanisms within glia that contribute to neurodegenerative disorders.
Minor weaknesses:
In Figure 1, the authors observe that in the cKO mice, the OPCs are dying because they observe a lack of NG2 staining. Is it possible the OPCs have changed to another cell identity that is NG2- in the absence of TDP-43? Tunnel staining would clarify that indeed the cKO OPCs are dying. Furthermore, the authors note that despite the extensive death of OPCs, they do not see signs of GFAP+ astrogliosis. Is there instead an increase in microglia activation? Throughout the paper, the authors use only GFAP+ astrogliosis to measure widespread inflammation. It would be more compelling to also look at the contribution of microglia or other inflammatory markers to measure inflammation.
As shown in Figure 3, loss of TDP-43 in oligodendrocytes at early and mature stages leads to similar profound phenotypes within both the Mobp-TDP43KO and Mogp-TDP43KO mouse lines. However, only early when TDP-43 is deleted using the Mobp-TDP43KO, are there severe physical phenotypes in the mice and early lethality. However, the authors show that there is no change in the density of ASPA+ mature oligodendrocytes in Mobp-TDP43KO and Mogp-TDP43KO at any stage. If there is an increase in the turnover of oligodendrocytes and oligodendrocyte number stays the same, can the authors speculate in their discussion what they believe is causing the severe seizure and lethality phenotypes in the Mobp-TDP43 KO mice? The authors mention that there is an increase in astrogliosis. Are they suggesting this change in astrocyte activity could promote the severe phenotypes and early lethality? Because motor neuron number is not affected by TDP-43 deletion, but no direct measurements of motor neuron activity were taken, it is hard to make sense of the phenotypes observed.
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