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Spliceosome integrity is defective in the motor neuron diseases ALS and SMA.

Tsuiji H, Iguchi Y, Furuya A, Kataoka A, Hatsuta H, Atsuta N, Tanaka F, Hashizume Y, Akatsu H, Murayama S, Sobue G, Yamanaka K - EMBO Mol Med (2013)

Bottom Line: We also show that in ALS, Gems are lost, U snRNA levels are up-regulated and spliceosomal U snRNPs abnormally and extensively accumulate in motor neuron nuclei, but not in the temporal lobe of FTLD with TDP-43 pathology.This aberrant accumulation of U snRNAs in ALS motor neurons is in direct contrast to SMA motor neurons, which show reduced amounts of U snRNAs, while both have defects in the spliceosome.These findings indicate that a profound loss of spliceosome integrity is a critical mechanism common to neurodegeneration in ALS and SMA, and may explain cell-type specific vulnerability of motor neurons.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Motor Neuron Disease, RIKEN Brain Science Institute, Wako, Saitama, Japan. hitomitsuiji@brain.riken.jp

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TDP-43 and FUS/TLS interact with the SMN complex in nuclear Gems, and are required for Gem formationA–C. Hela cells were immunostained with antibodies against TDP-43 and nuclear domain markers. Magnified images showing colocalization of TDP-43 and nuclear markers (upper right). (A,B) Costaining of TDP-43 and components of Gems. TDP-43 was extensively concentrated in Gems marked by SMN (A, arrows) or Gemin8 (B, arrows). (C) Costaining of Coilin, a Cajal body marker, and TDP-43. TDP-43 was concentrated in Cajal bodies marked by Coilin (arrows). Bars: 10 µm.D,E. TDP-43 localized in Gems of neuronal cell line SH-SY5Y (D, arrows) and primary cultured mouse hippocampal neurons (E, arrows). Bars: 10 µm.F. Costaining of FUS/TLS and Gemin8 in primary cultured mouse hippocampal neurons. FUS/TLS localized in Gem (arrows). Bars: 10 µm.G. Hela cells were treated with siRNAs for TDP-43 or control to deplete TDP-43, and immunostained for SMN and TDP-43. Gems are lost in cells with no TDP-43 expression (arrows), whereas they remain in cells with low TDP-43 expression (arrowheads). Bars: 10 µm.H. Quantification of Gem numbers in Hela cells treated with siRNAs shown in G. Cells with no TDP-43 expression in immunostaining were shown as knockdown (KD), whereas cells with low TDP-43 expression level in immunostaining were shown as mild KD. Means for number of Gems are 2.857 (Control, n = 21), 2.947 (mild KD, n = 19) and 0.1 (KD, n = 20) (Control vs KD: p < 0.0001).I. DIV 21 hippocampal neurons form FUS−/− mice or littermates were stained for SMN to analyze the requirement of FUS/TLS for Gem formation. Bars: 10 µm.J. Quantification of Gems positive for SMN. Means for number of Gems are 0.4026 (FUS+/+, n = 77) and 0.02667 (FUS−/−, n = 75) (p < 0.0001).
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fig01: TDP-43 and FUS/TLS interact with the SMN complex in nuclear Gems, and are required for Gem formationA–C. Hela cells were immunostained with antibodies against TDP-43 and nuclear domain markers. Magnified images showing colocalization of TDP-43 and nuclear markers (upper right). (A,B) Costaining of TDP-43 and components of Gems. TDP-43 was extensively concentrated in Gems marked by SMN (A, arrows) or Gemin8 (B, arrows). (C) Costaining of Coilin, a Cajal body marker, and TDP-43. TDP-43 was concentrated in Cajal bodies marked by Coilin (arrows). Bars: 10 µm.D,E. TDP-43 localized in Gems of neuronal cell line SH-SY5Y (D, arrows) and primary cultured mouse hippocampal neurons (E, arrows). Bars: 10 µm.F. Costaining of FUS/TLS and Gemin8 in primary cultured mouse hippocampal neurons. FUS/TLS localized in Gem (arrows). Bars: 10 µm.G. Hela cells were treated with siRNAs for TDP-43 or control to deplete TDP-43, and immunostained for SMN and TDP-43. Gems are lost in cells with no TDP-43 expression (arrows), whereas they remain in cells with low TDP-43 expression (arrowheads). Bars: 10 µm.H. Quantification of Gem numbers in Hela cells treated with siRNAs shown in G. Cells with no TDP-43 expression in immunostaining were shown as knockdown (KD), whereas cells with low TDP-43 expression level in immunostaining were shown as mild KD. Means for number of Gems are 2.857 (Control, n = 21), 2.947 (mild KD, n = 19) and 0.1 (KD, n = 20) (Control vs KD: p < 0.0001).I. DIV 21 hippocampal neurons form FUS−/− mice or littermates were stained for SMN to analyze the requirement of FUS/TLS for Gem formation. Bars: 10 µm.J. Quantification of Gems positive for SMN. Means for number of Gems are 0.4026 (FUS+/+, n = 77) and 0.02667 (FUS−/−, n = 75) (p < 0.0001).

Mentions: TDP-43 is an RNA-binding protein that predominantly localizes to the nucleus and regulates pre-mRNA splicing together with SR proteins or with other RNA-binding proteins such as hnRNPA2. The regulation of pre-mRNA is spatially and temporally controlled by splicing factors such as SR proteins that concentrate in nuclear speckles (Kumaran et al, 2008), suggesting that TDP-43 localizes to nuclear speckles as well as nucleoplasm. However, sub-nuclear localization of TDP-43 has been debated (Casafont et al, 2009; Fiesel et al, 2010; Shan et al, 2010; Wang et al, 2002). Our detailed analysis of sub-nuclear TDP-43 distribution revealed that TDP-43 was concentrated in Gems, which are marked by survival of motor neuron (SMN; Fig 1A, arrows) or Gemin8 (Fig 1B, arrows); Cajal bodies, which are marked by Coilin (Fig 1C, arrows); and paraspeckles, which are marked by p54nrb (Supporting Information Fig S1A, arrows). TDP-43 was partially overlapped with the SR protein SRSF2/SC35 in nuclear speckles (Supporting Information Fig S1B, arrows), and was not concentrated in PML bodies, the nucleolus, or SAM bodies (Supporting Information Fig S1C–E). TDP-43 was also localized to Gems in the neuronal cell line SH-SY5Y and in primary cultured neurons from mouse hippocampus (Fig 1D and E). FUS/TLS, an ALS-causative protein, was also localized to Gems in Hela cells (data not shown) and in primary cultured neurons from mouse hippocampus (Fig 1F). This is consistent given the known interaction of TDP-43 and FUS. Therefore, it is evident that both TDP-43 and FUS, RNA binding proteins of which mutations cause ALS, colocalize to Gems along with SMN.


Spliceosome integrity is defective in the motor neuron diseases ALS and SMA.

Tsuiji H, Iguchi Y, Furuya A, Kataoka A, Hatsuta H, Atsuta N, Tanaka F, Hashizume Y, Akatsu H, Murayama S, Sobue G, Yamanaka K - EMBO Mol Med (2013)

TDP-43 and FUS/TLS interact with the SMN complex in nuclear Gems, and are required for Gem formationA–C. Hela cells were immunostained with antibodies against TDP-43 and nuclear domain markers. Magnified images showing colocalization of TDP-43 and nuclear markers (upper right). (A,B) Costaining of TDP-43 and components of Gems. TDP-43 was extensively concentrated in Gems marked by SMN (A, arrows) or Gemin8 (B, arrows). (C) Costaining of Coilin, a Cajal body marker, and TDP-43. TDP-43 was concentrated in Cajal bodies marked by Coilin (arrows). Bars: 10 µm.D,E. TDP-43 localized in Gems of neuronal cell line SH-SY5Y (D, arrows) and primary cultured mouse hippocampal neurons (E, arrows). Bars: 10 µm.F. Costaining of FUS/TLS and Gemin8 in primary cultured mouse hippocampal neurons. FUS/TLS localized in Gem (arrows). Bars: 10 µm.G. Hela cells were treated with siRNAs for TDP-43 or control to deplete TDP-43, and immunostained for SMN and TDP-43. Gems are lost in cells with no TDP-43 expression (arrows), whereas they remain in cells with low TDP-43 expression (arrowheads). Bars: 10 µm.H. Quantification of Gem numbers in Hela cells treated with siRNAs shown in G. Cells with no TDP-43 expression in immunostaining were shown as knockdown (KD), whereas cells with low TDP-43 expression level in immunostaining were shown as mild KD. Means for number of Gems are 2.857 (Control, n = 21), 2.947 (mild KD, n = 19) and 0.1 (KD, n = 20) (Control vs KD: p < 0.0001).I. DIV 21 hippocampal neurons form FUS−/− mice or littermates were stained for SMN to analyze the requirement of FUS/TLS for Gem formation. Bars: 10 µm.J. Quantification of Gems positive for SMN. Means for number of Gems are 0.4026 (FUS+/+, n = 77) and 0.02667 (FUS−/−, n = 75) (p < 0.0001).
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fig01: TDP-43 and FUS/TLS interact with the SMN complex in nuclear Gems, and are required for Gem formationA–C. Hela cells were immunostained with antibodies against TDP-43 and nuclear domain markers. Magnified images showing colocalization of TDP-43 and nuclear markers (upper right). (A,B) Costaining of TDP-43 and components of Gems. TDP-43 was extensively concentrated in Gems marked by SMN (A, arrows) or Gemin8 (B, arrows). (C) Costaining of Coilin, a Cajal body marker, and TDP-43. TDP-43 was concentrated in Cajal bodies marked by Coilin (arrows). Bars: 10 µm.D,E. TDP-43 localized in Gems of neuronal cell line SH-SY5Y (D, arrows) and primary cultured mouse hippocampal neurons (E, arrows). Bars: 10 µm.F. Costaining of FUS/TLS and Gemin8 in primary cultured mouse hippocampal neurons. FUS/TLS localized in Gem (arrows). Bars: 10 µm.G. Hela cells were treated with siRNAs for TDP-43 or control to deplete TDP-43, and immunostained for SMN and TDP-43. Gems are lost in cells with no TDP-43 expression (arrows), whereas they remain in cells with low TDP-43 expression (arrowheads). Bars: 10 µm.H. Quantification of Gem numbers in Hela cells treated with siRNAs shown in G. Cells with no TDP-43 expression in immunostaining were shown as knockdown (KD), whereas cells with low TDP-43 expression level in immunostaining were shown as mild KD. Means for number of Gems are 2.857 (Control, n = 21), 2.947 (mild KD, n = 19) and 0.1 (KD, n = 20) (Control vs KD: p < 0.0001).I. DIV 21 hippocampal neurons form FUS−/− mice or littermates were stained for SMN to analyze the requirement of FUS/TLS for Gem formation. Bars: 10 µm.J. Quantification of Gems positive for SMN. Means for number of Gems are 0.4026 (FUS+/+, n = 77) and 0.02667 (FUS−/−, n = 75) (p < 0.0001).
Mentions: TDP-43 is an RNA-binding protein that predominantly localizes to the nucleus and regulates pre-mRNA splicing together with SR proteins or with other RNA-binding proteins such as hnRNPA2. The regulation of pre-mRNA is spatially and temporally controlled by splicing factors such as SR proteins that concentrate in nuclear speckles (Kumaran et al, 2008), suggesting that TDP-43 localizes to nuclear speckles as well as nucleoplasm. However, sub-nuclear localization of TDP-43 has been debated (Casafont et al, 2009; Fiesel et al, 2010; Shan et al, 2010; Wang et al, 2002). Our detailed analysis of sub-nuclear TDP-43 distribution revealed that TDP-43 was concentrated in Gems, which are marked by survival of motor neuron (SMN; Fig 1A, arrows) or Gemin8 (Fig 1B, arrows); Cajal bodies, which are marked by Coilin (Fig 1C, arrows); and paraspeckles, which are marked by p54nrb (Supporting Information Fig S1A, arrows). TDP-43 was partially overlapped with the SR protein SRSF2/SC35 in nuclear speckles (Supporting Information Fig S1B, arrows), and was not concentrated in PML bodies, the nucleolus, or SAM bodies (Supporting Information Fig S1C–E). TDP-43 was also localized to Gems in the neuronal cell line SH-SY5Y and in primary cultured neurons from mouse hippocampus (Fig 1D and E). FUS/TLS, an ALS-causative protein, was also localized to Gems in Hela cells (data not shown) and in primary cultured neurons from mouse hippocampus (Fig 1F). This is consistent given the known interaction of TDP-43 and FUS. Therefore, it is evident that both TDP-43 and FUS, RNA binding proteins of which mutations cause ALS, colocalize to Gems along with SMN.

Bottom Line: We also show that in ALS, Gems are lost, U snRNA levels are up-regulated and spliceosomal U snRNPs abnormally and extensively accumulate in motor neuron nuclei, but not in the temporal lobe of FTLD with TDP-43 pathology.This aberrant accumulation of U snRNAs in ALS motor neurons is in direct contrast to SMA motor neurons, which show reduced amounts of U snRNAs, while both have defects in the spliceosome.These findings indicate that a profound loss of spliceosome integrity is a critical mechanism common to neurodegeneration in ALS and SMA, and may explain cell-type specific vulnerability of motor neurons.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Motor Neuron Disease, RIKEN Brain Science Institute, Wako, Saitama, Japan. hitomitsuiji@brain.riken.jp

Show MeSH
Related in: MedlinePlus