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Background:
TDP-43 proteinopathy is a pathological hallmark of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). So far, there is no therapy available for these neurodegenerative diseases. In addition, the impact of TDP-43 proteinopathy on neuronal translational profile also remains unknown.
Methods:
Biochemical, immunohistology and assay-based studies were done with cell cultures and transgenic mice models. We also used Ribotag with microarray and proteomic analysis to determine the neuronal translational profile in the mice model of ALS/FTD.
Results:
Here, we report that oral administration of a novel analog (IMS-088) of withaferin-A, an antagonist of nuclear factor kappa-B (NF-ĸB) essential modulator (NEMO), induced autophagy and reduced TDP-43 proteinopathy in the brain and spinal cord of transgenic mice expressing human TDP-43 mutants, models of ALS/FTD. Treatment with IMS-088 ameliorated cognitive impairment, reduced gliosis in the brain of ALS/FTD mouse models. With the Ribotrap method, we investigated the impact of TDP-43 proteinopathy and IMS-088 treatment on the translation profile of neurons of one-year old hTDP-43A315T mice. TDP-43 proteinopathy caused translational dysregulation of specific mRNAs including translational suppression of neurofilament mRNAs resulting in 3 to 4-fold decrease in levels type IV neurofilament proteins. Oral administration of IMS-088 rescued the translational defects associated with TDP-43 proteinopathy and restored the synthesis of neurofilament proteins, which are essential for axon integrity and synaptic function.
Conclusions:
Our study revealed that induction of autophagy reduces TDP-43 pathology and ameliorates the translational defect seen in mice models of ALS/FTD. Based on these results, we suggest IMS-088 and perhaps other inducers of autophagy should be considered as potential therapeutics for neurodegenerative disorders with TDP-43 proteinopathies.
Keywords
Amyotrophic lateral sclerosis, fronto-temporal dementia, TDP-43, withaferin-A, autophagy, neurofilament, mRNA translation
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
This is a list of supplementary files associated with this preprint. Click to download.
- Fig.S1.tif
Figure S1 IMS-088 reduced levels of phospho-TDP43 in the brain of hTDP-43A315T mice. The anti-phospho-TDP-43 antibody (pSer410, Sigma-Aldrich, USA) was used for immunodetection (n=3 independent experiments).
- Fig.S2.tif
Figure S2 Inhibition of autophagy blocks IMS-088-mediated reduction of hTDP-43 aggregates. Immunoblots ad quantification of RIPA insoluble (A) and soluble (B) TDP-43 in HEK293 cells treated with ethacrynic acid with or without IMS-088 and Bafilmycin A1. HEK 293 cells were treated for 3 hrs with 50 µM Ethacrynic acid in serum free media and then with Bafilomycin A1 (300 nM) with or without IMS-088 for 6hrs. Post-treatment, levels of RIPA insoluble (A) or soluble (B) hTDP-43 in Hek293 cells were determined by immunoblotting with anti-human TDP-43 antibody (Abnova). (n=3; 1-way ANOVA with Bonferroni’s multiple comparison test as the post-test). Graphs show mean ± sem.
- Fig.S3.tif
Figure S3 (A) Schematic representation of HA-mRFP1-tagged murine Rpl10a ribosomal protein construct under control of the NFL 848 promoter to make neuron specific expression (B) Representative sketch showing the process used for generating double transgenic mice (by breeding NFL-RFP1 mice with mhTDP-43A315T mice) (C) Representative image showing DAPI (blue), NeuN a neuronal marker and RFP (red) in the brain of hTDP-43A315T mice and NfL-RFP;hTDP-43A315T double transgenic mice. (D) Representative image of experimental protocol used for the Ribotrap experiment.
- Fig.S4.tif
Figure S4 (A) Representative data of upregulated peptide associated GO biological function in NFLrRFP;hTDP-43A315T mice in comparison to NFLrRFP mice (B) Representative data of upregulated peptide associated GO biological function in NFLrRFP;hTDP-43A315T mice treated with IMS-088 in comparison to saline treatment. (C) Pie chart showing the majority of mRNA dysregulated after IMS-088 treatment found in Affymetrix Mouse Genome 430 analysis were pseudo or non-regulated genes with unknown function (D) Representative data of altered peptide including Pin1 in NFL-RFP; hTDP-43A315T and the impact of IMS-088 treatment on the reversal of neuronal translational profile in NFL-RFP; hTDP-43A315T mice.
- TableS1.tif
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CURRENT STATUS: Published
View the published article at Molecular Neurodegeneration.
Version 2
Posted 02 Dec, 2020
No community comments so far
Editorial decision: Accept
On 05 Dec, 2020
Review #2 received
Received 30 Nov, 2020
Reviewer #2 agreed
On 22 Nov, 2020
Review #1 received
Received 22 Nov, 2020
Reviewer #1 agreed
On 19 Nov, 2020
Editor assigned
On 18 Nov, 2020
Reviewers invited
Invitations sent on 18 Nov, 2020
Submission checks complete
On 18 Nov, 2020
Editor invited
On 18 Nov, 2020
No comments provided
Editorial decision: Major revision
On 03 Sep, 2020
Review #2 received
Received 21 Aug, 2020
Review #1 received
Received 10 Jun, 2020
Reviewer #2 agreed
On 08 Jun, 2020
Reviewer #1 agreed
On 02 Jun, 2020
Reviewers invited
Invitations sent on 01 Jun, 2020
Editor assigned
On 18 May, 2020
Submission checks complete
On 17 May, 2020
Editor invited
On 17 May, 2020
First submitted
On 15 May, 2020
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A new preprint design is coming!
We have improved readability, clarity, accessibility, and opportunities for engagement.
See the published version of this preprint at Molecular Neurodegeneration.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Molecular Neurodegeneration.
Version 2
Posted 02 Dec, 2020
No community comments so far
Editorial decision: Accept
On 05 Dec, 2020
Review #2 received
Received 30 Nov, 2020
Reviewer #2 agreed
On 22 Nov, 2020
Review #1 received
Received 22 Nov, 2020
Reviewer #1 agreed
On 19 Nov, 2020
Editor assigned
On 18 Nov, 2020
Reviewers invited
Invitations sent on 18 Nov, 2020
Submission checks complete
On 18 Nov, 2020
Editor invited
On 18 Nov, 2020
No comments provided
Editorial decision: Major revision
On 03 Sep, 2020
Review #2 received
Received 21 Aug, 2020
Review #1 received
Received 10 Jun, 2020
Reviewer #2 agreed
On 08 Jun, 2020
Reviewer #1 agreed
On 02 Jun, 2020
Reviewers invited
Invitations sent on 01 Jun, 2020
Editor assigned
On 18 May, 2020
Submission checks complete
On 17 May, 2020
Editor invited
On 17 May, 2020
First submitted
On 15 May, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Molecular Neurodegeneration.
Background:
TDP-43 proteinopathy is a pathological hallmark of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). So far, there is no therapy available for these neurodegenerative diseases. In addition, the impact of TDP-43 proteinopathy on neuronal translational profile also remains unknown.
Methods:
Biochemical, immunohistology and assay-based studies were done with cell cultures and transgenic mice models. We also used Ribotag with microarray and proteomic analysis to determine the neuronal translational profile in the mice model of ALS/FTD.
Results:
Here, we report that oral administration of a novel analog (IMS-088) of withaferin-A, an antagonist of nuclear factor kappa-B (NF-ĸB) essential modulator (NEMO), induced autophagy and reduced TDP-43 proteinopathy in the brain and spinal cord of transgenic mice expressing human TDP-43 mutants, models of ALS/FTD. Treatment with IMS-088 ameliorated cognitive impairment, reduced gliosis in the brain of ALS/FTD mouse models. With the Ribotrap method, we investigated the impact of TDP-43 proteinopathy and IMS-088 treatment on the translation profile of neurons of one-year old hTDP-43A315T mice. TDP-43 proteinopathy caused translational dysregulation of specific mRNAs including translational suppression of neurofilament mRNAs resulting in 3 to 4-fold decrease in levels type IV neurofilament proteins. Oral administration of IMS-088 rescued the translational defects associated with TDP-43 proteinopathy and restored the synthesis of neurofilament proteins, which are essential for axon integrity and synaptic function.
Conclusions:
Our study revealed that induction of autophagy reduces TDP-43 pathology and ameliorates the translational defect seen in mice models of ALS/FTD. Based on these results, we suggest IMS-088 and perhaps other inducers of autophagy should be considered as potential therapeutics for neurodegenerative disorders with TDP-43 proteinopathies.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
This is a list of supplementary files associated with this preprint. Click to download.
- Fig.S1.tif
Figure S1 IMS-088 reduced levels of phospho-TDP43 in the brain of hTDP-43A315T mice. The anti-phospho-TDP-43 antibody (pSer410, Sigma-Aldrich, USA) was used for immunodetection (n=3 independent experiments).
- Fig.S2.tif
Figure S2 Inhibition of autophagy blocks IMS-088-mediated reduction of hTDP-43 aggregates. Immunoblots ad quantification of RIPA insoluble (A) and soluble (B) TDP-43 in HEK293 cells treated with ethacrynic acid with or without IMS-088 and Bafilmycin A1. HEK 293 cells were treated for 3 hrs with 50 µM Ethacrynic acid in serum free media and then with Bafilomycin A1 (300 nM) with or without IMS-088 for 6hrs. Post-treatment, levels of RIPA insoluble (A) or soluble (B) hTDP-43 in Hek293 cells were determined by immunoblotting with anti-human TDP-43 antibody (Abnova). (n=3; 1-way ANOVA with Bonferroni’s multiple comparison test as the post-test). Graphs show mean ± sem.
- Fig.S3.tif
Figure S3 (A) Schematic representation of HA-mRFP1-tagged murine Rpl10a ribosomal protein construct under control of the NFL 848 promoter to make neuron specific expression (B) Representative sketch showing the process used for generating double transgenic mice (by breeding NFL-RFP1 mice with mhTDP-43A315T mice) (C) Representative image showing DAPI (blue), NeuN a neuronal marker and RFP (red) in the brain of hTDP-43A315T mice and NfL-RFP;hTDP-43A315T double transgenic mice. (D) Representative image of experimental protocol used for the Ribotrap experiment.
- Fig.S4.tif
Figure S4 (A) Representative data of upregulated peptide associated GO biological function in NFLrRFP;hTDP-43A315T mice in comparison to NFLrRFP mice (B) Representative data of upregulated peptide associated GO biological function in NFLrRFP;hTDP-43A315T mice treated with IMS-088 in comparison to saline treatment. (C) Pie chart showing the majority of mRNA dysregulated after IMS-088 treatment found in Affymetrix Mouse Genome 430 analysis were pseudo or non-regulated genes with unknown function (D) Representative data of altered peptide including Pin1 in NFL-RFP; hTDP-43A315T and the impact of IMS-088 treatment on the reversal of neuronal translational profile in NFL-RFP; hTDP-43A315T mice.
- TableS1.tif
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