TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS methods
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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.
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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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"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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