TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS methods
Aim. Evidence-backed execution summary for TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS methods from TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS.
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Cell transfection
reagent used in the protocol.
- Use
- Transfection of MEFs was performed using FuGENE HD (Promega) at a transfection reagent:DNA ratio of 3:1 for 48 hours. For HEK293T cells, Lipofectamine 2000 (Life Technologies) was used for 24 hours according to manufacturer's instructions.
TDP-43 Triggers mtDNA Release into the Cytoplasm
reagent used in the protocol.
- Use
- In sterile settings, cGAS can respond to double-stranded DNA (dsDNA) of either mitochondrial ( ) or nuclear origin ( ). To ascertain the source of DNA activating cGAS in response to TDP-43, we immunoprecipitated FLAG-tagged cGAS from HEK293T cells overexpressing WT or ALS mutant TDP-43 (A315T or Q331K; A), directly...
TDP-43 Entry into Mitochondria Requires the Translocase Subunit AGK
reagent used in the protocol.
- Use
- (E) Treatment with the TDP-43 inhibitor peptide (PM1; 1 µM for 24 h) prevents induction of IFNB1 and TNF in TDP-43-ALS patient iPSC-MNs.
TDP-43 Entry into Mitochondria Requires the Translocase Subunit AGK
reagent used in the protocol.
- Use
- (A) Representative western blot analysis of GFP-tagged TDP-43 (WT, A315T and Q331K), AGK, TFAM and Actin in Flp-In control, AGK -/-, AGK -/- +WT or AGK -/- +G126E HEK293T cells. (B) Representative western blot analysis of cells in panel (A), to establish the purity of D...
TDP-43 Triggers mtDNA Release into the Cytoplasm via the mPTP
reagent used in the protocol.
- Use
- After confirming the mode of entry of TDP-43 into mitochondria, we sought to identify the mechanism for mtDNA release. It has been reported that TDP-43 is capable of inducing apoptosis under certain conditions, which could trigger Bak/Bax permeabilization of the outer mitochondrial membrane and leakage of DNA into t...
Genetic Deletion of Sting Mitigates Disease in an ALS Mouse Model
reagent used in the protocol.
- Use
- To establish whether the cGAS/STING pathway is responsible for neuroinflammation in response to aberrant TDP-43 in vivo, we used the well-described murine model of ALS with human TDP-43 (p.A315T) overexpression in mice (referred to as Prp-TDP-43 Tg/+ ) ( ). When given access to a jellified diet, this strain av...
Genetic Deletion of Sting Mitigates Disease in an ALS Mouse Model
reagent used in the protocol.
- Use
- (A) qPCR analysis of type I IFNs ( Ifnb1 and Ifna6 ), interferon-stimulated genes ( Mx1, Ifit1 and Irf7 ) and NF-κB genes ( Tnf, Il6 and Il1b ) are presented for WT and Prp-TDP-43 Tg/+ bone marrow derived macrophages, taken from mice at 150 days of age. The mRNA expression was normalized to Hprt as relat...
Genetic Deletion of Sting Mitigates Disease in an ALS Mouse Model
reagent used in the protocol.
- Use
- (A) Quantification of cGAMP in the cortex, spinal cord, and serum of WT mice and mice that are transgenic for the human TDP-43 mutant allele A315T (Prp-TDP-43 Tg/+ ) (n = 5) at the experimental endpoint. See animal phenotype scoring.
TDP-43 Triggers mtDNA Release into the Cytoplasm
Video S1. TDP-43 Triggers mtDNA Release into the Cytoplasm, Related to Figure 2 Control MEFs or those overexpressing TDP-43 (WT or Q331K) were stained for TDP-43 (FLAG-tagged, red), mitochondrial inner membrane (TIM44, blue), mitochondrial outer membrane (TOM20, gray) and DNA (anti-DNA, green) then imaged by OM...
- Use
- Video S1. TDP-43 Triggers mtDNA Release into the Cytoplasm, Related to Figure 2 Control MEFs or those overexpressing TDP-43 (WT or Q331K) were stained for TDP-43 (FLAG-tagged, red), mitochondrial inner membrane (TIM44, blue), mitochondrial outer membrane (TOM20, gray) and DNA (anti-DNA, green) then imaged by OM...
TDP-43 Triggers mtDNA Release into the Cytoplasm via the mPTP
(D) Spatial quantification by Imaris software for the percentage of DNA outside of mitochondria (TIM44); 30-40 cells per group.
- Use
- (D) Spatial quantification by Imaris software for the percentage of DNA outside of mitochondria (TIM44); 30-40 cells per group.
TDP-43 Triggers mtDNA Release into the Cytoplasm
(D) OMX-SR microscopy reveals that TDP-43 (FLAG-tagged, red) translocates into mitochondria (TIM44, blue; TOM20, cyan) and induces relocation of DNA (anti-DNA, green) into the cytoplasm of TDP-43-overexpressing MEFs (scale bars, 5 µm). Overview images are maximum-intensity projections, and magnified images...
- Use
- (D) OMX-SR microscopy reveals that TDP-43 (FLAG-tagged, red) translocates into mitochondria (TIM44, blue; TOM20, cyan) and induces relocation of DNA (anti-DNA, green) into the cytoplasm of TDP-43-overexpressing MEFs (scale bars, 5 µm). Overview images are maximum-intensity projections, and magnified images...
TDP-43 Triggers mtDNA Release into the Cytoplasm
(E and F) Spatial quantification by Imaris software for (E) the percentage of FLAG-TDP-43 in mitochondria (TIM44) and (F) the percentage of DNA outside of mitochondria; 30-40 cells per group.
- Use
- (E and F) Spatial quantification by Imaris software for (E) the percentage of FLAG-TDP-43 in mitochondria (TIM44) and (F) the percentage of DNA outside of mitochondria; 30-40 cells per group.
TDP-43 Entry into Mitochondria Requires the Translocase Subunit AGK
(A) OMX-SR microscopy reveals that import of TDP-43 (Myc-tagged, red) into mitochondria (TIM44, blue) and TDP-43-induced relocation of DNA (anti-DNA, green) into the cytoplasm are ablated in HEK293T cells lacking the TIM22 regulatory subunit AGK (scale bars, 0.5 µm). Overview images are maximum-intensity p...
- Use
- (A) OMX-SR microscopy reveals that import of TDP-43 (Myc-tagged, red) into mitochondria (TIM44, blue) and TDP-43-induced relocation of DNA (anti-DNA, green) into the cytoplasm are ablated in HEK293T cells lacking the TIM22 regulatory subunit AGK (scale bars, 0.5 µm). Overview images are maximum-intensity p...
TDP-43 Entry into Mitochondria Requires the Translocase Subunit AGK
(B and C) Spatial quantification by Imaris software for (B) the percentage of Myc-TDP-43 in mitochondria (TIM44) and (C) the percentage of DNA outside of mitochondria in control, AGK -/-, AGK -/- +WT, or AGK -/- +G126E HEK293T cells; 30 cells per group.
- Use
- (B and C) Spatial quantification by Imaris software for (B) the percentage of Myc-TDP-43 in mitochondria (TIM44) and (C) the percentage of DNA outside of mitochondria in control, AGK -/-, AGK -/- +WT, or AGK -/- +G126E HEK293T cells; 30 cells per group.
TDP-43 Entry into Mitochondria Requires the Translocase Subunit AGK
Video S2. TDP-43 Import into Mitochondria Dependent on AGK, Related to Figure 3 HEK293T Flp-In control, AGK -/-, AGK -/- +WT or AGK -/- +G126E cells expressing TDP-43 (Q331K) were stained for TDP-43 (FLAG-tagged, red), mitochondrial inner membrane (TIM44, blue) and...
- Use
- Video S2. TDP-43 Import into Mitochondria Dependent on AGK, Related to Figure 3 HEK293T Flp-In control, AGK -/-, AGK -/- +WT or AGK -/- +G126E cells expressing TDP-43 (Q331K) were stained for TDP-43 (FLAG-tagged, red), mitochondrial inner membrane (TIM44, blue) and...
TDP-43 Triggers mtDNA Release into the Cytoplasm via the mPTP
(A) Representative western blot of cleaved caspase-3 in TDP-43-overexpressing Mcl1 -/- MEFs 72hrs post doxycycline (Dox) treatment, or treated with ABT-737 to induce apoptosis (t = 4h). (B) Representative western blot of Bak, Bax, TDP-43 and actin from cells in A. (C) iPSC-derived MNs from healthy c...
- Use
- (A) Representative western blot of cleaved caspase-3 in TDP-43-overexpressing Mcl1 -/- MEFs 72hrs post doxycycline (Dox) treatment, or treated with ABT-737 to induce apoptosis (t = 4h). (B) Representative western blot of Bak, Bax, TDP-43 and actin from cells in A. (C) iPSC-derived MNs from healthy c...
Genetic Deletion of Sting Mitigates Disease in an ALS Mouse Model
Software used for acquisition, scoring, statistics, or reporting.
- Use
- (A) qPCR analysis of type I IFNs ( Ifnb1 and Ifna6 ), interferon-stimulated genes ( Mx1, Ifit1 and Irf7 ) and NF-κB genes ( Tnf, Il6 and Il1b ) are presented for WT and Prp-TDP-43 Tg/+ bone marrow derived macrophages, taken from mice at 150 days of age. The mRNA expression was normalized to Hprt as relat...
Quantification and Statistical Analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Data are typically mean ± SEM and analyzed by t test between two groups or one- or two-way ANOVA followed by a Sidak, Tukey or Dunnett multiple comparison test as appropriate. GraphPad Prism 8 was used to generate all charts and statistical analyses. A P value < 0.05 was considered significant.
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Cell transfection
Transfection of MEFs was performed using FuGENE HD (Promega) at a transfection reagent:DNA ratio of 3:1 for 48 hours. For HEK293T cells, Lipofectamine 2000 (Life Technologies) was used for 24 hours according to manufacturer's instructions.
TDP-43 Triggers mtDNA Release into the Cytoplasm
In sterile settings, cGAS can respond to double-stranded DNA (dsDNA) of either mitochondrial ( ) or nuclear origin ( ). To ascertain the source of DNA activating cGAS in response to TDP-43, we immunoprecipitated FLAG-tagged cGAS from HEK293T cells overexpressing WT or ALS mutant TDP-43 (A315T or Q331K; A), directly followed by qPCR for the detection of mitochondrial or nuclear DNA ( ). This showed that, in response to TDP-43 overexpression, cGAS was bound to mitochondrial DNA (mtDNA) without evidence of the most abundant nuclear DNA sequences such as LINE1 elements or the ribosomal DNA RNA18S ( A). It is also possible to generate cell lines deficient for mtDNA (referred to as ρ 0 ) by culturing them with low-dose ethidium bromide (EtBr) ( ). mtDNA depletion from THP-1 cells containing inducible WT and mutant TDP-43 was achieved in culture with EtBr for 3 weeks, as judged by...
Human induced pluripotent stem cell (iPSC)
The established human iPSC lines used in this study were derived from six controls, three patients carrying mutations in TARDBP (G298S, M337V and A382T), three patients with repeat expansions in C9ORF72 and three patients carrying mutations in SOD1 (G85S, G93A and I114T). All iPSCs were maintained on Matrigel-coated 6-well plates in mTeSR1 containing 1x Primocin and passaged 1:6 using ReLeSR with ROCK inhibitor Y-27632 for the first 24 hours at 37°C in a humidified atmosphere with 5% CO 2. The medium was replaced daily. Cells were cyropreserved in mTeSR1/10% DMSO. See the for further information of the iPSC lines used in this study.
Mitochondrial DNA-depleted cells (ρ 0 )
Depletion of mtDNA was performed in a relevant culture medium containing 100 ng/mL ethidium bromide, 100 µg/mL sodium pyruvate and 50 µg/mL uridine as previously demonstrated ( ). THP-1 cells were cultured in this medium for 3 weeks and then rested for an additional 2 weeks in the presence of uridine to achieve mtDNA-depleted cells. Human iPSC-derived motor neurons were treated in a similar way, except a lower dose of 50 ng/mL ethidium bromide was used. The depletion was analyed using real-time qPCR to measure expression of mitochondrial genes or nuclear genes. The primer sequences used are provided in the.
CRISPR/Cas9-mediated gene deletion
We generated STING CRISPR-/- THP-1 cells and Ppid CRISPR-/- MEFs as described previously ( ). Third generation lentiviral transduction was performed to generate cells expressing Cas9 fused to mCherry or blasticidin ressitance gene, which were subjected to positive selection via FACS sorting or antibiotic treatment for 2 weeks respectively. Doxycycline-inducible sgRNAs were cloned using a pFgH1tUT (BFP tagged) plasmid and subsequently transduced into the target cells expressing Cas9. Cells were treated with doxycycline for 72 hours and then rested for an additional 48 hours prior to experiments. Gene disruption was confirmed by immunoblot analysis of target proteins and functional analysis. The targeting guide sequences are provided in the. Gene deletion was then confirmed by western blot and functional analysis.
Method Details
Third generation lentiviral constructs including pSLIK-Neo (vector), hTDP-43 WT and Q331K were used to generate lentivirus as described (; ). HEK293T cells were transiently transfected with pSLIK plasmids, pMDL (packaging), RSV-REV (packaging) and VSVg (envelope) using Lipofectamine 2000 diluted in OptiMEM (Thermo Fisher Scientific) to generate lentiviral particles. The cell culture supernatant was collected 48 hours later and filtured through 0.45 mm filteres prior to transduction, for which 5x10 5 target cells were centrifuged with the lentivirus in the presence of polybrene (Sigma-Aldrich) at 839 x g for 3 hours at 32°C and cultured at 37°C overnight. Transduced cells were subsequently subjected to antibiotic selection with G418 (Thermo Fisher Scientific) to generate stable cell lines carrying doxycycline-inducable 3xFLAG-tagged (N') and Myc-tagged (C')...
Generation of iPSC-derived motor neuron progenitors (MNPs)
Differentiation of iPSCs into MNPs was performed as described previously ( ). Human iPSCs were cultured in a chemically defined Neural Medium: DMEM/F12:Neurobasal (1:1) supplemented with 0.5x N-2, 0.5x B-27, 0.1mM L-ascorbic acid, 1x Glutamax and 1x Primocin containing 3 µM CHIR99021, 2 µM Dorsomorphin and 2 µM SB431542 37°C in a humidified atmosphere with 5% CO 2 for 6 days for induction of neuroepithelial progenitors (NEPs). The NEPs were dissociated with ReLeSR as per manufacturer's instruction and cultured 1:6 on Matrigel-coated plates in the Neural Medium containing 1 µM CHIR99021, 2 µM Dorsomorphin and 2 µM SB431542 for 6 days for induction of MNPs. Y-27632 was used for the first 24 hours, and the medium was changed every other day. At this stage of differentiation, MNPs were either expanded in the...
Differentiation of motor neurons (MNs)
The MNPs were dissociated with Accutase and cultured on Matrigel-coated plates in the Neural Medium containing 0.5 µM all-trans RA and 0.1 µM Pur for 6 days into premature MNX1 + MN. Y-27632 was used for the first 24 hours. Subsequently, cells were detached with Accutase to generate a single cell suspension and matured in the medium supplemented with 0.1 µM Compound E for 10 days into ChAT + MNs. The medium was replaced every other day for both stages of differentiation. Cell markers of motor neurons, including MNX1, ChAT and βIII-Tubulin, were confirmed using an inverted SP8 confocal microscopy (Leica) or quantitative real-time PCR. The antibodies and primers used are provided in the.
Measurement outputs
What raw and processed outputs should exist?
(E and F) Inhibition of the mPTP (CsA, 12.5 µM) in HEK293T cells prevents mtDNA cytosolic accumulation (cytosolic/total lysis, percent) and (F) prevents Ifnb1 gene exp...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
(G and H) CRISPR-mediated genetic deletion of the mPTP component Ppid also abolished mtDNA release into the cytoplasm and (H) downstream Ifnb1 gene expression.
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
(F) qPCR of inflammatory gene expression relative to Hprt in the cortex and spinal cord reveals that increased levels of type I IFN- and NF-κB-dependent cytokines are great...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
In sterile settings, cGAS can respond to double-stranded DNA (dsDNA) of either mitochondrial ( ) or nuclear origin ( ). To ascertain the source of DNA activating cGAS in respons...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
All animals studied here were males.
from paperScoring or quantification
Quantify the primary readouts for this experiment: (E and F) Inhibition of the mPTP (CsA, 12.5 µM) in HEK293T cells prevents mtDNA cytosolic accumulation (cytosolic/total lysis, percent) and (F) prevents Ifnb1 gene exp...; (G and H) CRISPR-mediated genetic deletion of the mPTP component Ppid also abolished mtDNA release into the cytoplasm and (H) downstream Ifnb1 gene expression.; (F) qPCR of inflammatory gene expression relative to Hprt in the cortex and spinal cord reveals that increased levels of type I IFN- and NF-κB-dependent cytokines are great...; In sterile settings, cGAS can respond to double-stranded DNA (dsDNA) of either mitochondrial ( ) or nuclear origin ( ). To ascertain the source of DNA activating cGAS in respons....
from paperStatistical comparison
All animals studied here were males. Data are mean ± SEM. The p values were calculated using unpaired t test between two groups in (A) or one-way ANOVA to Prp-TDP-43 Tg/+ S...; Data are mean - SEM, pooled from 3 independent experiments ([A], [B], [E], and [F]) or representative of 3 independent experiments ([C] and [D]). The p values were calcula...; In sterile settings, cGAS can respond to double-stranded DNA (dsDNA) of either mitochondrial ( ) or nuclear origin ( ). To ascertain the source of DNA activating cGAS in respons...; (A) Representative western blot of FLAG-cGAS immunoprecipitation from A. (B) Representative qPCR analysis of mitochondrial DNA (mtDNA) depletion from THP-1 cells over three week...
from paperReporting output
Report representative outputs alongside summary comparisons for (E and F) Inhibition of the mPTP (CsA, 12.5 µM) in HEK293T cells prevents mtDNA cytosolic accumulation (cytosolic/total lysis, percent) and (F) prevents Ifnb1 gene exp..., (G and H) CRISPR-mediated genetic deletion of the mPTP component Ppid also abolished mtDNA release into the cytoplasm and (H) downstream Ifnb1 gene expression., (F) qPCR of inflammatory gene expression relative to Hprt in the cortex and spinal cord reveals that increased levels of type I IFN- and NF-κB-dependent cytokines are great..., In sterile settings, cGAS can respond to double-stranded DNA (dsDNA) of either mitochondrial ( ) or nuclear origin ( ). To ascertain the source of DNA activating cGAS in respons....
inferred from protocolStructured statistical methods
All animals studied here were males. Data are mean ± SEM. The p values were calculated using unpaired t test between two groups in (A) or one-way ANOVA to Prp-TDP-43 Tg/+ S...; Data are mean - SEM, pooled from 3 independent experiments ([A], [B], [E], and [F]) or representative of 3 independent experiments ([C] and [D]). The p values were calcula...; In sterile settings, cGAS can respond to double-stranded DNA (dsDNA) of either mitochondrial ( ) or nuclear origin ( ). To ascertain the source of DNA activating cGAS in respons...; (A) Representative western blot of FLAG-cGAS immunoprecipitation from A. (B) Representative qPCR analysis of mitochondrial DNA (mtDNA) depletion from THP-1 cells over three week...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Transfection of MEFs was performed using FuGENE HD (Promega) at a transfection reagent:DNA ratio of 3:1 for 48 hours. For HEK293T cells, Lipofectamine 2000 (Life Technologies) was used for 24 hours according to manufacturer's instructions.
In sterile settings, cGAS can respond to double-stranded DNA (dsDNA) of either mitochondrial ( ) or nuclear origin ( ). To ascertain the source of DNA activating cGAS in response to TDP-43, we immunoprecipitated FLAG-tagged cGAS from HEK293T cells overexpressing WT or ALS mutant TDP-43 (A315T or Q331K; A), directly followed by qPCR for the detection of mitochondrial or nuclear DNA ( ). This showed that, in response to TDP-43 overexpression, cGAS was bound to mitochondrial DNA (mtDNA) without evidence of the most abundant nuclear DNA sequences such as LINE1 elements or the ribosomal DNA RNA18S ( A). It is also possible to generate cell lines deficient for mtDNA (referred to as ρ 0 ) by culturing them with low-dose ethidium bromide (EtBr) ( ). mtDNA depletion from THP-1 cells containing inducible WT and mutant TDP-43 was achieved in culture with EtBr for 3 weeks, as judged by qPCR of mitochondrial gene expression ( B). We then treated the cells with doxycycline (Dox) to induce TDP-43 (WT or Q331K), followed by quantification of inflammatory cytokine expression ( B), and activation of signaling pathways downstream of cGAS/STING ( C), which returned to baseline in the `...
The established human iPSC lines used in this study were derived from six controls, three patients carrying mutations in TARDBP (G298S, M337V and A382T), three patients with repeat expansions in C9ORF72 and three patients carrying mutations in SOD1 (G85S, G93A and I114T). All iPSCs were maintained on Matrigel-coated 6-well plates in mTeSR1 containing 1x Primocin and passaged 1:6 using ReLeSR with ROCK inhibitor Y-27632 for the first 24 hours at 37°C in a humidified atmosphere with 5% CO 2. The medium was replaced daily. Cells were cyropreserved in mTeSR1/10% DMSO. See the for further information of the iPSC lines used in this study.
Depletion of mtDNA was performed in a relevant culture medium containing 100 ng/mL ethidium bromide, 100 µg/mL sodium pyruvate and 50 µg/mL uridine as previously demonstrated ( ). THP-1 cells were cultured in this medium for 3 weeks and then rested for an additional 2 weeks in the presence of uridine to achieve mtDNA-depleted cells. Human iPSC-derived motor neurons were treated in a similar way, except a lower dose of 50 ng/mL ethidium bromide was used. The depletion was analyed using real-time qPCR to measure expression of mitochondrial genes or nuclear genes. The primer sequences used are provided in the.
We generated STING CRISPR-/- THP-1 cells and Ppid CRISPR-/- MEFs as described previously ( ). Third generation lentiviral transduction was performed to generate cells expressing Cas9 fused to mCherry or blasticidin ressitance gene, which were subjected to positive selection via FACS sorting or antibiotic treatment for 2 weeks respectively. Doxycycline-inducible sgRNAs were cloned using a pFgH1tUT (BFP tagged) plasmid and subsequently transduced into the target cells expressing Cas9. Cells were treated with doxycycline for 72 hours and then rested for an additional 48 hours prior to experiments. Gene disruption was confirmed by immunoblot analysis of target proteins and functional analysis. The targeting guide sequences are provided in the. Gene deletion was then confirmed by western blot and functional analysis.
Third generation lentiviral constructs including pSLIK-Neo (vector), hTDP-43 WT and Q331K were used to generate lentivirus as described (; ). HEK293T cells were transiently transfected with pSLIK plasmids, pMDL (packaging), RSV-REV (packaging) and VSVg (envelope) using Lipofectamine 2000 diluted in OptiMEM (Thermo Fisher Scientific) to generate lentiviral particles. The cell culture supernatant was collected 48 hours later and filtured through 0.45 mm filteres prior to transduction, for which 5x10 5 target cells were centrifuged with the lentivirus in the presence of polybrene (Sigma-Aldrich) at 839 x g for 3 hours at 32°C and cultured at 37°C overnight. Transduced cells were subsequently subjected to antibiotic selection with G418 (Thermo Fisher Scientific) to generate stable cell lines carrying doxycycline-inducable 3xFLAG-tagged (N') and Myc-tagged (C') TDP-43.
Differentiation of iPSCs into MNPs was performed as described previously ( ). Human iPSCs were cultured in a chemically defined Neural Medium: DMEM/F12:Neurobasal (1:1) supplemented with 0.5x N-2, 0.5x B-27, 0.1mM L-ascorbic acid, 1x Glutamax and 1x Primocin containing 3 µM CHIR99021, 2 µM Dorsomorphin and 2 µM SB431542 37°C in a humidified atmosphere with 5% CO 2 for 6 days for induction of neuroepithelial progenitors (NEPs). The NEPs were dissociated with ReLeSR as per manufacturer's instruction and cultured 1:6 on Matrigel-coated plates in the Neural Medium containing 1 µM CHIR99021, 2 µM Dorsomorphin and 2 µM SB431542 for 6 days for induction of MNPs. Y-27632 was used for the first 24 hours, and the medium was changed every other day. At this stage of differentiation, MNPs were either expanded in the Neural Medium containing 3 µM CHIR99021, 2 µM Dorsomorphin, 2 µM SB431542, 0.1 µM all-trans retinoic acid (RA), 0.5 µM Purmorphamine (Pur) and 0.5 mM Valproic Acid prior to differentiation of motor neuron or cryopreserved in the same medium cont...
The MNPs were dissociated with Accutase and cultured on Matrigel-coated plates in the Neural Medium containing 0.5 µM all-trans RA and 0.1 µM Pur for 6 days into premature MNX1 + MN. Y-27632 was used for the first 24 hours. Subsequently, cells were detached with Accutase to generate a single cell suspension and matured in the medium supplemented with 0.1 µM Compound E for 10 days into ChAT + MNs. The medium was replaced every other day for both stages of differentiation. Cell markers of motor neurons, including MNX1, ChAT and βIII-Tubulin, were confirmed using an inverted SP8 confocal microscopy (Leica) or quantitative real-time PCR. The antibodies and primers used are provided in the.
Machine-readable layer
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"name": "TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS methods",
"description": "Evidence-backed execution summary for TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS methods from TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS.",
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"text": "Transfection of MEFs was performed using FuGENE HD (Promega) at a transfection reagent:DNA ratio of 3:1 for 48 hours. For HEK293T cells, Lipofectamine 2000 (Life Technologies) was used for 24 hours according to manufacturer's instructions."
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"name": "TDP-43 Triggers mtDNA Release into the Cytoplasm",
"text": "In sterile settings, cGAS can respond to double-stranded DNA (dsDNA) of either mitochondrial ( ) or nuclear origin ( ). To ascertain the source of DNA activating cGAS in response to TDP-43, we immunoprecipitated FLAG-tagged cGAS from HEK293T cells overexpressing WT or ALS mutant TDP-43 (A315T or Q331K; A), directly followed by qPCR for the detection of mitochondrial or nuclear DNA ( ). This showed that, in response to TDP-43 overexpression, cGAS was bound to mitochondrial DNA (mtDNA) without evidence of the most abundant nuclear DNA sequences such as LINE1 elements or the ribosomal DNA RNA18S ( A). It is also possible to generate cell lines deficient for mtDNA (referred to as ρ 0 ) by culturing them with low-dose ethidium bromide (EtBr) ( ). mtDNA depletion from THP-1 cells containing inducible WT and mutant TDP-43 was achieved in culture with EtBr for 3 weeks, as judged by..."
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"name": "Human induced pluripotent stem cell (iPSC)",
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"name": "Mitochondrial DNA-depleted cells (ρ 0 )",
"text": "Depletion of mtDNA was performed in a relevant culture medium containing 100 ng/mL ethidium bromide, 100 µg/mL sodium pyruvate and 50 µg/mL uridine as previously demonstrated ( ). THP-1 cells were cultured in this medium for 3 weeks and then rested for an additional 2 weeks in the presence of uridine to achieve mtDNA-depleted cells. Human iPSC-derived motor neurons were treated in a similar way, except a lower dose of 50 ng/mL ethidium bromide was used. The depletion was analyed using real-time qPCR to measure expression of mitochondrial genes or nuclear genes. The primer sequences used are provided in the."
},
{
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"name": "CRISPR/Cas9-mediated gene deletion",
"text": "We generated STING CRISPR-/- THP-1 cells and Ppid CRISPR-/- MEFs as described previously ( ). Third generation lentiviral transduction was performed to generate cells expressing Cas9 fused to mCherry or blasticidin ressitance gene, which were subjected to positive selection via FACS sorting or antibiotic treatment for 2 weeks respectively. Doxycycline-inducible sgRNAs were cloned using a pFgH1tUT (BFP tagged) plasmid and subsequently transduced into the target cells expressing Cas9. Cells were treated with doxycycline for 72 hours and then rested for an additional 48 hours prior to experiments. Gene disruption was confirmed by immunoblot analysis of target proteins and functional analysis. The targeting guide sequences are provided in the. Gene deletion was then confirmed by western blot and functional analysis."
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"text": "Third generation lentiviral constructs including pSLIK-Neo (vector), hTDP-43 WT and Q331K were used to generate lentivirus as described (; ). HEK293T cells were transiently transfected with pSLIK plasmids, pMDL (packaging), RSV-REV (packaging) and VSVg (envelope) using Lipofectamine 2000 diluted in OptiMEM (Thermo Fisher Scientific) to generate lentiviral particles. The cell culture supernatant was collected 48 hours later and filtured through 0.45 mm filteres prior to transduction, for which 5x10 5 target cells were centrifuged with the lentivirus in the presence of polybrene (Sigma-Aldrich) at 839 x g for 3 hours at 32°C and cultured at 37°C overnight. Transduced cells were subsequently subjected to antibiotic selection with G418 (Thermo Fisher Scientific) to generate stable cell lines carrying doxycycline-inducable 3xFLAG-tagged (N') and Myc-tagged (C')..."
},
{
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"position": 7,
"name": "Generation of iPSC-derived motor neuron progenitors (MNPs)",
"text": "Differentiation of iPSCs into MNPs was performed as described previously ( ). Human iPSCs were cultured in a chemically defined Neural Medium: DMEM/F12:Neurobasal (1:1) supplemented with 0.5x N-2, 0.5x B-27, 0.1mM L-ascorbic acid, 1x Glutamax and 1x Primocin containing 3 µM CHIR99021, 2 µM Dorsomorphin and 2 µM SB431542 37°C in a humidified atmosphere with 5% CO 2 for 6 days for induction of neuroepithelial progenitors (NEPs). The NEPs were dissociated with ReLeSR as per manufacturer's instruction and cultured 1:6 on Matrigel-coated plates in the Neural Medium containing 1 µM CHIR99021, 2 µM Dorsomorphin and 2 µM SB431542 for 6 days for induction of MNPs. Y-27632 was used for the first 24 hours, and the medium was changed every other day. At this stage of differentiation, MNPs were either expanded in the..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Differentiation of motor neurons (MNs)",
"text": "The MNPs were dissociated with Accutase and cultured on Matrigel-coated plates in the Neural Medium containing 0.5 µM all-trans RA and 0.1 µM Pur for 6 days into premature MNX1 + MN. Y-27632 was used for the first 24 hours. Subsequently, cells were detached with Accutase to generate a single cell suspension and matured in the medium supplemented with 0.1 µM Compound E for 10 days into ChAT + MNs. The medium was replaced every other day for both stages of differentiation. Cell markers of motor neurons, including MNX1, ChAT and βIII-Tubulin, were confirmed using an inverted SP8 confocal microscopy (Leica) or quantitative real-time PCR. The antibodies and primers used are provided in the."
}
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"name": "Genetic Deletion of Sting Mitigates Disease in an ALS Mouse Model"
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"name": "Genetic Deletion of Sting Mitigates Disease in an ALS Mouse Model"
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{
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"datePublished": "2020",
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