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HDAC6 inhibition restores TDP‐43 pathology and axonal transport defects in human motor neurons with TARDBP mutations

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Fazal, Raheem, Boeynaems, Steven, Swijsen, Ann, De Decker, Mathias, Fumagalli, Laura, Moisse, Matthieu, Vanneste, Joni, Guo, Wenting, Boon, Ruben, Vercruysse, Thomas, Eggermont, Kristel, Swinnen, Bart, Beckers, Jimmy, Pakravan, Donya, Vandoorne, Tijs, Vanden Berghe, Pieter, Verfaillie, Catherine, Van Den Bosch, Ludo, Van Damme, Philip·2021·The EMBO Journal
DOI 10.15252/embj.2020106177PMC PMC8013789
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Abstract

TDP‐43 is the major component of pathological inclusions in most ALS patients and in up to 50% of patients with frontotemporal dementia (FTD). Heterozygous missense mutations in TARDBP , the gene encoding TDP‐43, are one of the common causes of familial ALS. In this study, we investigate TDP‐43 protein behavior in induced pluripotent stem cell (iPSC)‐derived motor neurons from three ALS patients with different TARDBP mutations, three healthy controls and an isogenic control. TARDPB mutations induce several TDP‐43 changes in spinal motor neurons, including cytoplasmic mislocalization and accumulation of insoluble TDP‐43, C‐terminal fragments, and phospho‐TDP‐43. By generating iPSC lines with allele‐specific tagging of TDP‐43, we find that mutant TDP‐43 initiates the observed disease phenotypes and has an altered interactome as indicated by mass spectrometry. Our findings also indicate that TDP‐43 proteinopathy results in a defect in mitochondrial transport. Lastly, we show that pharmacological inhibition of histone deacetylase 6 (HDAC6) restores the observed TDP‐43 pathologies and the axonal mitochondrial motility, suggesting that HDAC6 inhibition may be an interesting therapeutic target for neurodegenerative disorders linked to TDP‐43 pathology.

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Figures

Figure 1 Increased levels of insoluble TDP‐43 and C‐terminal TDP‐43 fragments in mutant TDP‐43 motor neurons AScheme representing the workflow of fractionation into soluble and insoluble fractions.B, C(B) Representative Western blot showing total TDP‐43 levels (unfractionated), quantification in (C), ratio paired t‐test.DWestern blot analysis of soluble and insoluble fractions (Red and yellow rectangles highlight CTF‐35 and CTF‐25, respectively, in the insoluble fraction).EQuantification of soluble full‐length TDP‐43, ratio paired t‐test.FQuantification of insoluble full‐length TDP‐43, ratio paired t‐test.GGraph contains quantification of insoluble c‐terminal fragment of 35 kDa (CTF‐35), ratio paired t‐test.HGraph indicates quantification of insoluble c‐terminal fragment of 25 kDa (CTF‐25), ratio paired t‐test. Data information: Data are shown as mean ± SEM, *P < 0.05, **P < 0.01, ns: not significant. Each dot represents an independent differentiation in all panels: Data obtained from five‐six independent differentiations.
Figure 1 Increased levels of insoluble TDP‐43 and C‐terminal TDP‐43 fragments in mutant TDP‐43 motor neurons AScheme representing the workflow of fractionation into soluble and insoluble fractions.B, C(B) Representative Western blot showing total TDP‐43 levels (unfractionated), quantification in (C), ratio paired t‐test.DWestern blot analysis of soluble and insoluble fractions (Red and yellow rectangles highlight CTF‐35 and CTF‐25, respectively, in the insoluble fraction).EQuantification of soluble full‐length TDP‐43, ratio paired t‐test.FQuantification of insoluble full‐length TDP‐43, ratio paired t‐test.GGraph contains quantification of insoluble c‐terminal fragment of 35 kDa (CTF‐35), ratio paired t‐test.HGraph indicates quantification of insoluble c‐terminal fragment of 25 kDa (CTF‐25), ratio paired t‐test. Data information: Data are shown as mean ± SEM, *P < 0.05, **P < 0.01, ns: not significant. Each dot represents an independent differentiation in all panels: Data obtained from five‐six independent differentiations.CC BY · PubMed Central
Figure 2 Mutant TDP‐43 motor neurons exhibit TDP‐43 cytoplasmic mislocalization and increased phosphorylation AImmunofluorescent analysis of TDP‐43 in control and mutant TDP‐43 iPSC‐derived motor neurons. Upper images represent control and lower mutant TDP‐43 motor neurons. Presented immunofluorescent images of control (C2) and mutant TDP‐43 (P2), Scale bar: 20 µm.BCorresponding profile intensity plot of panel A.CQuantification of the nucleo‐cytoplasmic ratio (N/C) of TDP‐43 fluorescent intensity of pooled control and pooled mutant motor neurons, unpaired Mann–Whitney test.D, E(D) Western blot analysis of cytoplasmic fraction with a positive nuclear control (C) confirming the fractionation purity, quantification in (E), ratio paired t‐test.F, G(F) Western blot analysis of nuclear fraction with a positive cytoplasmic control (C) confirming the fractionation purity, quantification in (G), ratio paired t‐test.H–JImmunofluorescent analysis showing fluorescence intensity of phosphorylated TDP‐43 (pTDP‐43). Scale bar: 20 µm. Presented immunofluorescent images of control (C2) and mutant TDP‐43 (P2). (I) represents its corresponding profile intensity plot of the selected (square) region. Scale bar: 10 µm. (J) quantification pooled control (C0 = 10, C1 = 10, and C2 = 10) n = 30 and pooled mutant TDP‐43 (P1 = 10, P2 = 10, and P3 = 10) n = 30 cells from each differentiation. Unpaired Mann–Whitney test.K, L(K) Western blot analysis showing pTDP‐43, quantification in (L), ratio paired t‐test. Data information: Data are shown as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant. Each dot represents an independent differentiation in all panels: Data obtained from three independent differentiations.
Figure 2 Mutant TDP‐43 motor neurons exhibit TDP‐43 cytoplasmic mislocalization and increased phosphorylation AImmunofluorescent analysis of TDP‐43 in control and mutant TDP‐43 iPSC‐derived motor neurons. Upper images represent control and lower mutant TDP‐43 motor neurons. Presented immunofluorescent images of control (C2) and mutant TDP‐43 (P2), Scale bar: 20 µm.BCorresponding profile intensity plot of panel A.CQuantification of the nucleo‐cytoplasmic ratio (N/C) of TDP‐43 fluorescent intensity of pooled control and pooled mutant motor neurons, unpaired Mann–Whitney test.D, E(D) Western blot analysis of cytoplasmic fraction with a positive nuclear control (C) confirming the fractionation purity, quantification in (E), ratio paired t‐test.F, G(F) Western blot analysis of nuclear fraction with a positive cytoplasmic control (C) confirming the fractionation purity, quantification in (G), ratio paired t‐test.H–JImmunofluorescent analysis showing fluorescence intensity of phosphorylated TDP‐43 (pTDP‐43). Scale bar: 20 µm. Presented immunofluorescent images of control (C2) and mutant TDP‐43 (P2). (I) represents its corresponding profile intensity plot of the selected (square) region. Scale bar: 10 µm. (J) quantification pooled control (C0 = 10, C1 = 10, and C2 = 10) n = 30 and pooled mutant TDP‐43 (P1 = 10, P2 = 10, and P3 = 10) n = 30 cells from each differentiation. Unpaired Mann–Whitney test.K, L(K) Western blot analysis showing pTDP‐43, quantification in (L), ratio paired t‐test. Data information: Data are shown as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant. Each dot represents an independent differentiation in all panels: Data obtained from three independent differentiations.CC BY · PubMed Central
Figure 3 Mutant TDP‐43 undergoes cytoplasmic mislocalization and increased phosphorylation in mutant TDP‐43 motor neurons AScheme representing the two cell lines generated from the mutant TDP‐43 iPSCs by either endogenously tagging the WT TDP‐43 allele or the mutant TDP‐43 allele with mCherry.B, C(B) Immunofluorescent analysis with in (C) its corresponding intensity plot showing cytoplasmic mislocalization of the MUT‐mCherry TDP‐43 in motor neurons, but not in WT‐mCherry TDP‐43. Scale bar: 20 µm.DQuantification of nucleo‐cytoplasmic (N/C) intensity ratio based on immunohistochemistry of WT‐mCherry (n = 45) and MUT‐mCherry (n = 63) tagged TDP‐43. Each dot represents one analyzed cell, unpaired Mann–Whitney test.E, F(E) Western blots showing nucleo‐cytoplasmic fractionation of WT‐mCherry and MUT‐mCherry iPSC‐derived motor neurons with (F) (left panel) Quantification of total mCherry and total untagged TDP‐43 on α‐tubulin and (right panel) relative cytoplasmic mCherry and untagged TDP‐43 on total mCherry and total untagged TDP‐43, respectively, ratio paired t‐test.G(left panel) Quantification of total mCherry and total untagged TDP‐43 on Histone H4 and (right panel) relative nuclear mCherry and untagged TDP‐43 on total mCherry and total untagged TDP‐43, respectively, ratio paired t‐test.H, I(H) Western blot showing untagged TDP‐43 (left panel) and phosphorylation state of the untagged allele (right panel) in WT‐ and MUT‐mCherry motor neurons and in (I) ratio quantification of normalized pTDP43/normalized TDP‐43 (because of almost the same molecular weight two different blots were used), ratio paired t‐test. Data information: Data are shown as mean ± SEM, *P < 0.05, **P < 0.01, ****P < 0.0001. Each dot represents an independent differentiation in (F, G, and I) panels: Data obtained from three independent differentiations.
Figure 3 Mutant TDP‐43 undergoes cytoplasmic mislocalization and increased phosphorylation in mutant TDP‐43 motor neurons AScheme representing the two cell lines generated from the mutant TDP‐43 iPSCs by either endogenously tagging the WT TDP‐43 allele or the mutant TDP‐43 allele with mCherry.B, C(B) Immunofluorescent analysis with in (C) its corresponding intensity plot showing cytoplasmic mislocalization of the MUT‐mCherry TDP‐43 in motor neurons, but not in WT‐mCherry TDP‐43. Scale bar: 20 µm.DQuantification of nucleo‐cytoplasmic (N/C) intensity ratio based on immunohistochemistry of WT‐mCherry (n = 45) and MUT‐mCherry (n = 63) tagged TDP‐43. Each dot represents one analyzed cell, unpaired Mann–Whitney test.E, F(E) Western blots showing nucleo‐cytoplasmic fractionation of WT‐mCherry and MUT‐mCherry iPSC‐derived motor neurons with (F) (left panel) Quantification of total mCherry and total untagged TDP‐43 on α‐tubulin and (right panel) relative cytoplasmic mCherry and untagged TDP‐43 on total mCherry and total untagged TDP‐43, respectively, ratio paired t‐test.G(left panel) Quantification of total mCherry and total untagged TDP‐43 on Histone H4 and (right panel) relative nuclear mCherry and untagged TDP‐43 on total mCherry and total untagged TDP‐43, respectively, ratio paired t‐test.H, I(H) Western blot showing untagged TDP‐43 (left panel) and phosphorylation state of the untagged allele (right panel) in WT‐ and MUT‐mCherry motor neurons and in (I) ratio quantification of normalized pTDP43/normalized TDP‐43 (because of almost the same molecular weight two different blots were used), ratio paired t‐test. Data information: Data are shown as mean ± SEM, *P < 0.05, **P < 0.01, ****P < 0.0001. Each dot represents an independent differentiation in (F, G, and I) panels: Data obtained from three independent differentiations.CC BY · PubMed Central
Figure 4 Mutant TDP‐43 motor neurons display mitochondrial transport defects Representative kymographs from control and mutant TDP‐43 iPSC‐derived motor neurons. The x‐axis represents distance in µm while on the y‐axis time in seconds is revealed. Stationary mitochondria are visible as straight vertical lines, while moving mitochondria are depicted as skewed lines.Quantification of percentage of moving mitochondria.Quantification of the absolute number of moving mitochondria.Quantification of stationary mitochondria normalized to neurite length 100 µm. Data information: Data are shown as mean ± SEM, **P < 0.01, ****P < 0.0001. For panels (B, C, and D): Each dot represents one neurite for control (n = 81) and mutant TDP‐43 (n = 141) neurites. Mann–Whitney test in all panels. Data combined from three independent differentiations.
Figure 4 Mutant TDP‐43 motor neurons display mitochondrial transport defects Representative kymographs from control and mutant TDP‐43 iPSC‐derived motor neurons. The x‐axis represents distance in µm while on the y‐axis time in seconds is revealed. Stationary mitochondria are visible as straight vertical lines, while moving mitochondria are depicted as skewed lines.Quantification of percentage of moving mitochondria.Quantification of the absolute number of moving mitochondria.Quantification of stationary mitochondria normalized to neurite length 100 µm. Data information: Data are shown as mean ± SEM, **P < 0.01, ****P < 0.0001. For panels (B, C, and D): Each dot represents one neurite for control (n = 81) and mutant TDP‐43 (n = 141) neurites. Mann–Whitney test in all panels. Data combined from three independent differentiations.CC BY · PubMed Central
Figure 5 CRISPR/Cas9‐mediated gene editing reverse total and insoluble TDP‐43 AWestern blot analysis of total fraction of TDP‐43 levels in healthy controls (C0, C1, and C2), isogenic P1 (Iso‐P1), and mutant P1 (P1) motor neurons.BQuantification of total full‐length TDP‐43 levels, ratio paired t‐test.C, D(C) Western blot of soluble fraction and (D) quantification of soluble full‐length TDP‐43, ratio paired t‐test.E, F(E) Western blot of insoluble fraction and (F) quantification of insoluble full‐length TDP‐43, ratio paired t‐test.G, H(G) quantification of insoluble CTF‐35, ratio paired t‐test and (H) quantification of insoluble CTF‐25, ratio paired t‐test. Data information: Data are shown as mean ± SEM. *P < 0.05, ns: not significant. Each dot represents an independent differentiation in all panels: Data combined from three independent differentiations.
Figure 5 CRISPR/Cas9‐mediated gene editing reverse total and insoluble TDP‐43 AWestern blot analysis of total fraction of TDP‐43 levels in healthy controls (C0, C1, and C2), isogenic P1 (Iso‐P1), and mutant P1 (P1) motor neurons.BQuantification of total full‐length TDP‐43 levels, ratio paired t‐test.C, D(C) Western blot of soluble fraction and (D) quantification of soluble full‐length TDP‐43, ratio paired t‐test.E, F(E) Western blot of insoluble fraction and (F) quantification of insoluble full‐length TDP‐43, ratio paired t‐test.G, H(G) quantification of insoluble CTF‐35, ratio paired t‐test and (H) quantification of insoluble CTF‐25, ratio paired t‐test. Data information: Data are shown as mean ± SEM. *P < 0.05, ns: not significant. Each dot represents an independent differentiation in all panels: Data combined from three independent differentiations.CC BY · PubMed Central
Figure 6 CRISPR/Cas9‐mediated gene editing reverses mutant TDP‐43‐associated phenotypes AWestern blot analysis of cytoplasmic TDP‐43 protein levels in healthy controls (C0, C1, and C2), isogenic P1 (Iso‐P1), and mutant P1 (P1) motor neurons.BQuantification of cytoplasmic TDP‐43 levels, each dot represents an independent differentiation, ratio paired t‐test.C, D(C) Western blot analysis of nuclear TDP‐43 and (D) quantification of nuclear TDP‐43 levels, each dot represents an independent differentiation, ratio paired t‐test.E, F(E) Immunofluorescent analysis showing fluorescence intensity of pTDP‐43 in motor neurons. Scale bar: 10 µm and its perspective intensity plot in (F).GQuantification of (E), each dot represents one analyzed cell, unpaired Mann–Whitney test.H, I(H) Western blot analysis showing pTDP‐43 levels in healthy controls, Iso‐P1 and P1, quantification in (I), each dot represents one analyzed cell, unpaired Mann–Whitney test.J–LAxonal transport analysis. Quantification of percentage of moving mitochondria (J), number of moving mitochondria (K), and number of stationary mitochondria (L) normalized to neurite length 100 µm. Each dot represents one neurite for Iso‐P1 (n = 26) and P1 (n = 32) neurites. Unpaired Mann–Whitney test. Data information: Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, ns: not significant. Data combined from three independent differentiations.
Figure 6 CRISPR/Cas9‐mediated gene editing reverses mutant TDP‐43‐associated phenotypes AWestern blot analysis of cytoplasmic TDP‐43 protein levels in healthy controls (C0, C1, and C2), isogenic P1 (Iso‐P1), and mutant P1 (P1) motor neurons.BQuantification of cytoplasmic TDP‐43 levels, each dot represents an independent differentiation, ratio paired t‐test.C, D(C) Western blot analysis of nuclear TDP‐43 and (D) quantification of nuclear TDP‐43 levels, each dot represents an independent differentiation, ratio paired t‐test.E, F(E) Immunofluorescent analysis showing fluorescence intensity of pTDP‐43 in motor neurons. Scale bar: 10 µm and its perspective intensity plot in (F).GQuantification of (E), each dot represents one analyzed cell, unpaired Mann–Whitney test.H, I(H) Western blot analysis showing pTDP‐43 levels in healthy controls, Iso‐P1 and P1, quantification in (I), each dot represents one analyzed cell, unpaired Mann–Whitney test.J–LAxonal transport analysis. Quantification of percentage of moving mitochondria (J), number of moving mitochondria (K), and number of stationary mitochondria (L) normalized to neurite length 100 µm. Each dot represents one neurite for Iso‐P1 (n = 26) and P1 (n = 32) neurites. Unpaired Mann–Whitney test. Data information: Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, ns: not significant. Data combined from three independent differentiations.CC BY · PubMed Central
Figure 7 HDAC6 inhibitor reduces total TDP‐43 levels by reducing the insoluble TDP‐43 levels and c‐terminal fragmentations in mutant TDP‐43 iPSC‐derived motor neurons A–D(A) Western blot showing total TDP‐43 levels, quantification in (B), ratio paired t‐test. Total cell lysates were fractionated into soluble and insoluble fractions and analyzed by Western blot, representative Western blots of soluble and insoluble fraction without (C) and with (D) HDAC6 inhibitor treatment.E, F(E) Quantification of soluble full‐length TDP‐43 levels, ratio paired t‐test and (F) quantification of insoluble full‐length TDP‐43 levels, ratio paired t‐test.GWestern blots of insoluble fraction with (right) and without (left) HDAC6 inhibitor treatment.H, IQuantification of insoluble c‐terminal fragment of 35 kDa (CTF‐35; H) and insoluble c‐terminal fragment of 25 kDa (CTF‐25; I), ratio paired t‐test. Data information: Data are shown as mean ± SEM, *P < 0.1, **P < 0.01, ***P < 0.001, ns: not significant. Each dot represents an independent differentiation in all panels: Data combined from three independent differentiations.
Figure 7 HDAC6 inhibitor reduces total TDP‐43 levels by reducing the insoluble TDP‐43 levels and c‐terminal fragmentations in mutant TDP‐43 iPSC‐derived motor neurons A–D(A) Western blot showing total TDP‐43 levels, quantification in (B), ratio paired t‐test. Total cell lysates were fractionated into soluble and insoluble fractions and analyzed by Western blot, representative Western blots of soluble and insoluble fraction without (C) and with (D) HDAC6 inhibitor treatment.E, F(E) Quantification of soluble full‐length TDP‐43 levels, ratio paired t‐test and (F) quantification of insoluble full‐length TDP‐43 levels, ratio paired t‐test.GWestern blots of insoluble fraction with (right) and without (left) HDAC6 inhibitor treatment.H, IQuantification of insoluble c‐terminal fragment of 35 kDa (CTF‐35; H) and insoluble c‐terminal fragment of 25 kDa (CTF‐25; I), ratio paired t‐test. Data information: Data are shown as mean ± SEM, *P < 0.1, **P < 0.01, ***P < 0.001, ns: not significant. Each dot represents an independent differentiation in all panels: Data combined from three independent differentiations.CC BY · PubMed Central
Figure 8 HDAC6 inhibitor restores subcellular mislocalization and abnormal phosphorylation of TDP‐43 in mutant TDP‐43 iPSC‐derived motor neurons AImmunofluorescent analysis of TDP‐43 in control, mutant TDP‐43, and mutant TDP‐43 + HDAC6 inhibitor after 12 h treatment. Presented immunofluorescent images of control (C2) and mutant TDP‐43 (P2). Scale bar: 25 µm.BProfile intensity plot of control, mutant TDP‐43, and mutant TDP‐43 + HDAC6 inhibitor.CQuantification of nucleo‐cytoplasmic ratio (N/C) fluorescent intensity of TDP‐43 in motor neurons, each dot represents one analyzed cell, unpaired Mann–Whitney test.D, E(D) Western blot analysis of cytosolic fraction, quantification in (E), ratio paired t‐test.F, G(F) Western blot analysis of nuclear fraction, quantification in (G), ratio paired t‐test.H, I(H) Western blot analysis showing pTDP‐43 levels and (I) quantification, ratio paired t‐test. Data information: Data are shown as mean ± SEM, *P < 0.05, ***P < 0.001, ****P < 0.0001, ns: not significant. Each dot represents an independent differentiation in (E, G, and I) panels: Data combined from three independent differentiations.
Figure 8 HDAC6 inhibitor restores subcellular mislocalization and abnormal phosphorylation of TDP‐43 in mutant TDP‐43 iPSC‐derived motor neurons AImmunofluorescent analysis of TDP‐43 in control, mutant TDP‐43, and mutant TDP‐43 + HDAC6 inhibitor after 12 h treatment. Presented immunofluorescent images of control (C2) and mutant TDP‐43 (P2). Scale bar: 25 µm.BProfile intensity plot of control, mutant TDP‐43, and mutant TDP‐43 + HDAC6 inhibitor.CQuantification of nucleo‐cytoplasmic ratio (N/C) fluorescent intensity of TDP‐43 in motor neurons, each dot represents one analyzed cell, unpaired Mann–Whitney test.D, E(D) Western blot analysis of cytosolic fraction, quantification in (E), ratio paired t‐test.F, G(F) Western blot analysis of nuclear fraction, quantification in (G), ratio paired t‐test.H, I(H) Western blot analysis showing pTDP‐43 levels and (I) quantification, ratio paired t‐test. Data information: Data are shown as mean ± SEM, *P < 0.05, ***P < 0.001, ****P < 0.0001, ns: not significant. Each dot represents an independent differentiation in (E, G, and I) panels: Data combined from three independent differentiations.CC BY · PubMed Central
Figure 9 HDAC6 inhibitor reduces cytoplasmic MUT‐mCherry TDP‐43 and enhances nuclear WT‐mCherry TDP‐43 ARepresentative Western blot of total (mCherry and untagged), cytoplasmic TDP‐43 and nuclear levels with and without HDAC6 inhibitor.BQuantification of total mCherry and total untagged relative to α‐tubulin, ratio paired t‐test.CQuantification of relative cytoplasmic mCherry TDP‐43 on total fraction, ratio paired t‐test.DQuantification of total mCherry and total untagged relative to Histone H4, ratio paired t‐test.EQuantification of relative nuclear mCherry TDP‐43 on total fraction, ratio paired t‐test.F, G(F) Western blot showing untagged TDP‐43 (left panel) and phosphorylation state of the untagged allele (right panel) in WT‐ and MUT‐mCherry motor neurons and in (G) ratio quantification of normalized pTDP43/normalized TDP‐43 (because of almost the same molecular weight two different blots were used), ratio paired t‐test. Data information: Data are shown as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant. Each dot represents an independent differentiation in all panels: Data combined from three independent differentiations.
Figure 9 HDAC6 inhibitor reduces cytoplasmic MUT‐mCherry TDP‐43 and enhances nuclear WT‐mCherry TDP‐43 ARepresentative Western blot of total (mCherry and untagged), cytoplasmic TDP‐43 and nuclear levels with and without HDAC6 inhibitor.BQuantification of total mCherry and total untagged relative to α‐tubulin, ratio paired t‐test.CQuantification of relative cytoplasmic mCherry TDP‐43 on total fraction, ratio paired t‐test.DQuantification of total mCherry and total untagged relative to Histone H4, ratio paired t‐test.EQuantification of relative nuclear mCherry TDP‐43 on total fraction, ratio paired t‐test.F, G(F) Western blot showing untagged TDP‐43 (left panel) and phosphorylation state of the untagged allele (right panel) in WT‐ and MUT‐mCherry motor neurons and in (G) ratio quantification of normalized pTDP43/normalized TDP‐43 (because of almost the same molecular weight two different blots were used), ratio paired t‐test. Data information: Data are shown as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant. Each dot represents an independent differentiation in all panels: Data combined from three independent differentiations.CC BY · PubMed Central
Figure 10 HDAC6 inhibitor rescues mitochondrial transport defects in mutant TDP‐43 iPSC‐derived motor neurons A–C(A) Quantification of percentage of moving mitochondria, (B) quantification of the absolute amount of moving mitochondria, and (C) quantification of the amount of stationary mitochondria normalized to neurite length 100 µm in each quantification. Each dot represents one neurite for control (n = 94) versus mutant TDP‐43 (n = 159) and control treated with HDAC6 inhibitor (n = 145) versus mutant treated with HDAC6 inhibitor (n = 235). Mann–Whitney test in all panels: Data were combined from three independent differentiations. Data are shown as mean ± SEM, **P < 0.01, ****P < 0.0001, ns: not significant.
Figure 10 HDAC6 inhibitor rescues mitochondrial transport defects in mutant TDP‐43 iPSC‐derived motor neurons A–C(A) Quantification of percentage of moving mitochondria, (B) quantification of the absolute amount of moving mitochondria, and (C) quantification of the amount of stationary mitochondria normalized to neurite length 100 µm in each quantification. Each dot represents one neurite for control (n = 94) versus mutant TDP‐43 (n = 159) and control treated with HDAC6 inhibitor (n = 145) versus mutant treated with HDAC6 inhibitor (n = 235). Mann–Whitney test in all panels: Data were combined from three independent differentiations. Data are shown as mean ± SEM, **P < 0.01, ****P < 0.0001, ns: not significant.CC BY · PubMed Central
Figure 11 TDP‐43 interactome analysis highlights pathological pathways and therapeutic effect of HDAC6 inhibition AScheme illustrating set‐up of TDP‐43 interactome analysis.BRanked GO enrichment analysis of mutant versus wild‐type TDP‐43 interactors indicates nuclear depletion of mutant TDP‐43.CMore detailed GO analysis further highlights nuclear depletion of mutant TDP‐43 and potential loss‐of‐function mechanisms.DSeveral axonal transport factors differentially interact with mutant versus wild‐type TDP‐43.EThe mutant TDP‐43 interactome is depleted for nuclear transport factors and enriched for proteostasis factors, in line with its observed nuclear depletion and cytoplasmic pathological alterations.FScheme illustrating the effect of HDAC6 inhibition on mutant TDP‐43 phenotypes.G, HRanked GO enrichment analysis highlights the effect of HDAC6 inhibition on the mutant TDP‐43 interactome, in line with its restored nuclear localization.
Figure 11 TDP‐43 interactome analysis highlights pathological pathways and therapeutic effect of HDAC6 inhibition AScheme illustrating set‐up of TDP‐43 interactome analysis.BRanked GO enrichment analysis of mutant versus wild‐type TDP‐43 interactors indicates nuclear depletion of mutant TDP‐43.CMore detailed GO analysis further highlights nuclear depletion of mutant TDP‐43 and potential loss‐of‐function mechanisms.DSeveral axonal transport factors differentially interact with mutant versus wild‐type TDP‐43.EThe mutant TDP‐43 interactome is depleted for nuclear transport factors and enriched for proteostasis factors, in line with its observed nuclear depletion and cytoplasmic pathological alterations.FScheme illustrating the effect of HDAC6 inhibition on mutant TDP‐43 phenotypes.G, HRanked GO enrichment analysis highlights the effect of HDAC6 inhibition on the mutant TDP‐43 interactome, in line with its restored nuclear localization.CC BY · PubMed Central

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Research works printed but not linked: 2

  1. van Es MA (2017) Amyotrophic lateral sclerosis Overview & Practice Essentials. Medscape 377: 1602
  2. Soneson C, Love MI, Robinson MD (2016) Differential analyses for RNA‐seq: transcript‐level inferences. F1000Res 4: 1521 · DOI · Paper page

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Authors
Fazal, R., Boeynaems, S., Swijsen, A., De Decker, M., Fumagalli, L., Moisse, M., Vanneste, J., Guo, W., Boon, R., Vercruysse, T., Eggermont, K., Swinnen, B., Beckers, J., Pakravan, D., Vandoorne, T., Vanden Berghe, P., Verfaillie, C., Van Den Bosch, L., & Van Damme, P.
Affiliations
  • VIB-KU Leuven Center for Brain & Disease Research (BE)
  • Stanford Medicine (US)
  • Leuven Stem Cell Institute (BE)
  • Rega Institute for Medical Research (BE)
  • Universitair Ziekenhuis Leuven (BE)
ORCID
Venue
The EMBO Journal
Publisher
Springer Science and Business Media LLC
Published
2021-03-10
Volume
40
Issue
7
Pages
e106177-e106177
ISSN
  • 0261-4189 (ISSN-L)
  • 1460-2075
DOI
10.15252/embj.2020106177
PMC
PMC8013789
OpenAlex
W3133763219
Cited by
108
References
88
Citations by year
2026: 15 · 2025: 25 · 2024: 26 · 2023: 18 · 2022: 19 · 2021: 5
FWCI
8.10
Type
journal article
Language
en
Open access
Licence
Locations
  • The EMBO Journal (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Lirias (KU Leuven) (submittedVersion, other-oa)
  • Data Archiving and Networked Services (DANS) (submittedVersion, other-oa)
  • Pure Amsterdam UMC (submittedVersion, cc-by)
Funders
  • Onderzoeksraad, KU Leuven (C1 ‐ C14‐17‐107)
  • Katholieke Universiteit Leuven | Universitaire Ziekenhuizen Leuven, KU Leuven (C1 ‐ C14‐17‐107)
  • Alzheimer's Research Foundation (SAO‐FRA n{degree sign} 2017/023)
  • VIND (135043)
  • Fonds Wetenschappelijk Onderzoek (G.0909.15, G0A7418N)
  • Agentschap voor Innovatie door Wetenschap en Technologie (150031)
  • The european E‐Rare‐3 project INTEGRALS
  • The ALS Liga Belgie
  • KULeuven fund &quot;een hart voor ALS&quot;
  • Laeversfonds voor ALS onderzoek
  • Valery Perrier Race against ALS fund
  • National lottery Belgium
  • Target ALS
  • E. von Behring Chair for Neuromuscular Disorders
  • EMBO Long‐term Fellowship
Topics
  • Health Sciences › Medicine › Clinical Neurology › Amyotrophic Lateral Sclerosis Research
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Hereditary Neurological Disorders
Keywords
TDP-43, TARDBP mutations, HDAC6 inhibition, amyotrophic lateral sclerosis, motor neurons, axonal transport, mitochondrial transport, proteinopathies, TDP-43 mislocalization, protein-protein interaction network, iPSC-derived motor neurons, frontotemporal dementia, therapeutic target
MeSH terms
Histone Deacetylase 6, Amyotrophic Lateral Sclerosis, Axonal Transport, Cells, Cultured, DNA-Binding Proteins, Humans, Mitochondria, Motor Neurons, Mutation, Missense, Histone Deacetylase Inhibitors, Induced Pluripotent Stem Cells

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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