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A splicing mutation in the novel mitochondrial protein DNAJC11 causes motor neuron pathology associated with cristae disorganization, and lymphoid abnormalities in mice.

Ioakeimidis F, Ott C, Kozjak-Pavlovic V, Violitzi F, Rinotas V, Makrinou E, Eliopoulos E, Fasseas C, Kollias G, Douni E - PLoS ONE (2014)

Bottom Line: The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog.The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization.Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Agricultural University of Athens, Athens, Greece; Division of Immunology, Biomedical Sciences Research Center "Alexander Fleming", Vari, Greece.

ABSTRACT
Mitochondrial structure and function is emerging as a major contributor to neuromuscular disease, highlighting the need for the complete elucidation of the underlying molecular and pathophysiological mechanisms. Following a forward genetics approach with N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we identified a novel mouse model of autosomal recessive neuromuscular disease caused by a splice-site hypomorphic mutation in a novel gene of unknown function, DnaJC11. Recent findings have demonstrated that DNAJC11 protein co-immunoprecipitates with proteins of the mitochondrial contact site (MICOS) complex involved in the formation of mitochondrial cristae and cristae junctions. Homozygous mutant mice developed locomotion defects, muscle weakness, spasticity, limb tremor, leucopenia, thymic and splenic hypoplasia, general wasting and early lethality. Neuropathological analysis showed severe vacuolation of the motor neurons in the spinal cord, originating from dilatations of the endoplasmic reticulum and notably from mitochondria that had lost their proper inner membrane organization. The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog. The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization. Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells. Our findings provide the first mouse mutant for a putative MICOS protein and establish a link between DNAJC11 and neuromuscular diseases.

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Expression analysis of mutant muDNAJC11 and biochemical interaction of huDNAJC11 with MICOS members.(A) Western blot analysis of fractionated brain tissue from DnaJC11spc/spc (spc/spc) mice and WT (+/+) littermates showing the loss of muDNAJC11 protein in DnaJC11spc/spc tissue. Prohibitin is a mitochondrial specific marker and GAPDH a cytoplasmic marker. (B) Equal amounts of isolated mitochondria from the indicated mouse tissues were analyzed by Western blot and probed for known members of the MICOS complex. Glucose related protein 75 (GRP75) was used as a loading control. (C) dnajc11kd-3, (D) sam50kd-2 or (E) mflkd-2 cells were grown in the absence (-Dox) or presence (+Dox) of doxycycline for 7 or 14 days, mitochondria were isolated, and 25 or 50 µg of protein were analyzed by SDS-PAGE and probed for the indicated proteins. SDHA, the component of the respiratory complex II, was used as a loading control. CHCHD3 and 6, coiled-coil-helix-coiled-coil-helix domain containing protein 3 and 6; SAM50, Sorting and assembly machinery 50; SDHA, Succinate Dehydrogenase subunit A. (F) Mitochondria from non-induced and induced sam50kd-2 knockdown cells after 7 days of induction with doxycyclin (Dox) were isolated and incubated with the radiolabeled mitofilin and DNAJC11 (the longest isoform) for the indicated time periods. Samples were analyzed by BN-PAGE and autoradiography. The panel on the right hand side shows the control of the knockdown, where 50 µg of mitochondria from –Dox and +Dox samples were analyzed by SDS-PAGE and Western blot using antibodies against Sam50 and SDHA.
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pone-0104237-g007: Expression analysis of mutant muDNAJC11 and biochemical interaction of huDNAJC11 with MICOS members.(A) Western blot analysis of fractionated brain tissue from DnaJC11spc/spc (spc/spc) mice and WT (+/+) littermates showing the loss of muDNAJC11 protein in DnaJC11spc/spc tissue. Prohibitin is a mitochondrial specific marker and GAPDH a cytoplasmic marker. (B) Equal amounts of isolated mitochondria from the indicated mouse tissues were analyzed by Western blot and probed for known members of the MICOS complex. Glucose related protein 75 (GRP75) was used as a loading control. (C) dnajc11kd-3, (D) sam50kd-2 or (E) mflkd-2 cells were grown in the absence (-Dox) or presence (+Dox) of doxycycline for 7 or 14 days, mitochondria were isolated, and 25 or 50 µg of protein were analyzed by SDS-PAGE and probed for the indicated proteins. SDHA, the component of the respiratory complex II, was used as a loading control. CHCHD3 and 6, coiled-coil-helix-coiled-coil-helix domain containing protein 3 and 6; SAM50, Sorting and assembly machinery 50; SDHA, Succinate Dehydrogenase subunit A. (F) Mitochondria from non-induced and induced sam50kd-2 knockdown cells after 7 days of induction with doxycyclin (Dox) were isolated and incubated with the radiolabeled mitofilin and DNAJC11 (the longest isoform) for the indicated time periods. Samples were analyzed by BN-PAGE and autoradiography. The panel on the right hand side shows the control of the knockdown, where 50 µg of mitochondria from –Dox and +Dox samples were analyzed by SDS-PAGE and Western blot using antibodies against Sam50 and SDHA.

Mentions: The predicted replacement of the last 51 amino acids of the muDNAJC11 by 43 different ones could interfere with proper mitochondrial localization of the protein. Thus, in order to examine the subcellular localization and the tissue distribution of the DNAJC11spc mutant protein, Western blot analyses on cytosolic and mitochondrial fractions were performed in various tissues. Our results showed that in cerebrum and cerebellum of DnaJC11spc/spc mice the muDNAJC11 protein was not present in either fraction (Figure 7A). Additional Western blots on isolated mitochondria from various tissues of DnaJC11spc/spc mice revealed that the DnaJC11spc mutation resulted in a reduction of muDNAJC11 protein levels ranging from severe to complete depletion depending on the tissue (Figure 7B). Thus, it is suggested that the DnaJC11spc allele ranges from severely hypomorphic to depending on the tissue. The possibility that the observed muDNAJC11 bands could represent the predicted mutant form of the protein is open, though. The two proteins, WT and mutant, differ only in 1 kDa in mass and thus could not be resolved.


A splicing mutation in the novel mitochondrial protein DNAJC11 causes motor neuron pathology associated with cristae disorganization, and lymphoid abnormalities in mice.

Ioakeimidis F, Ott C, Kozjak-Pavlovic V, Violitzi F, Rinotas V, Makrinou E, Eliopoulos E, Fasseas C, Kollias G, Douni E - PLoS ONE (2014)

Expression analysis of mutant muDNAJC11 and biochemical interaction of huDNAJC11 with MICOS members.(A) Western blot analysis of fractionated brain tissue from DnaJC11spc/spc (spc/spc) mice and WT (+/+) littermates showing the loss of muDNAJC11 protein in DnaJC11spc/spc tissue. Prohibitin is a mitochondrial specific marker and GAPDH a cytoplasmic marker. (B) Equal amounts of isolated mitochondria from the indicated mouse tissues were analyzed by Western blot and probed for known members of the MICOS complex. Glucose related protein 75 (GRP75) was used as a loading control. (C) dnajc11kd-3, (D) sam50kd-2 or (E) mflkd-2 cells were grown in the absence (-Dox) or presence (+Dox) of doxycycline for 7 or 14 days, mitochondria were isolated, and 25 or 50 µg of protein were analyzed by SDS-PAGE and probed for the indicated proteins. SDHA, the component of the respiratory complex II, was used as a loading control. CHCHD3 and 6, coiled-coil-helix-coiled-coil-helix domain containing protein 3 and 6; SAM50, Sorting and assembly machinery 50; SDHA, Succinate Dehydrogenase subunit A. (F) Mitochondria from non-induced and induced sam50kd-2 knockdown cells after 7 days of induction with doxycyclin (Dox) were isolated and incubated with the radiolabeled mitofilin and DNAJC11 (the longest isoform) for the indicated time periods. Samples were analyzed by BN-PAGE and autoradiography. The panel on the right hand side shows the control of the knockdown, where 50 µg of mitochondria from –Dox and +Dox samples were analyzed by SDS-PAGE and Western blot using antibodies against Sam50 and SDHA.
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Related In: Results  -  Collection

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Show All Figures
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pone-0104237-g007: Expression analysis of mutant muDNAJC11 and biochemical interaction of huDNAJC11 with MICOS members.(A) Western blot analysis of fractionated brain tissue from DnaJC11spc/spc (spc/spc) mice and WT (+/+) littermates showing the loss of muDNAJC11 protein in DnaJC11spc/spc tissue. Prohibitin is a mitochondrial specific marker and GAPDH a cytoplasmic marker. (B) Equal amounts of isolated mitochondria from the indicated mouse tissues were analyzed by Western blot and probed for known members of the MICOS complex. Glucose related protein 75 (GRP75) was used as a loading control. (C) dnajc11kd-3, (D) sam50kd-2 or (E) mflkd-2 cells were grown in the absence (-Dox) or presence (+Dox) of doxycycline for 7 or 14 days, mitochondria were isolated, and 25 or 50 µg of protein were analyzed by SDS-PAGE and probed for the indicated proteins. SDHA, the component of the respiratory complex II, was used as a loading control. CHCHD3 and 6, coiled-coil-helix-coiled-coil-helix domain containing protein 3 and 6; SAM50, Sorting and assembly machinery 50; SDHA, Succinate Dehydrogenase subunit A. (F) Mitochondria from non-induced and induced sam50kd-2 knockdown cells after 7 days of induction with doxycyclin (Dox) were isolated and incubated with the radiolabeled mitofilin and DNAJC11 (the longest isoform) for the indicated time periods. Samples were analyzed by BN-PAGE and autoradiography. The panel on the right hand side shows the control of the knockdown, where 50 µg of mitochondria from –Dox and +Dox samples were analyzed by SDS-PAGE and Western blot using antibodies against Sam50 and SDHA.
Mentions: The predicted replacement of the last 51 amino acids of the muDNAJC11 by 43 different ones could interfere with proper mitochondrial localization of the protein. Thus, in order to examine the subcellular localization and the tissue distribution of the DNAJC11spc mutant protein, Western blot analyses on cytosolic and mitochondrial fractions were performed in various tissues. Our results showed that in cerebrum and cerebellum of DnaJC11spc/spc mice the muDNAJC11 protein was not present in either fraction (Figure 7A). Additional Western blots on isolated mitochondria from various tissues of DnaJC11spc/spc mice revealed that the DnaJC11spc mutation resulted in a reduction of muDNAJC11 protein levels ranging from severe to complete depletion depending on the tissue (Figure 7B). Thus, it is suggested that the DnaJC11spc allele ranges from severely hypomorphic to depending on the tissue. The possibility that the observed muDNAJC11 bands could represent the predicted mutant form of the protein is open, though. The two proteins, WT and mutant, differ only in 1 kDa in mass and thus could not be resolved.

Bottom Line: The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog.The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization.Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Agricultural University of Athens, Athens, Greece; Division of Immunology, Biomedical Sciences Research Center "Alexander Fleming", Vari, Greece.

ABSTRACT
Mitochondrial structure and function is emerging as a major contributor to neuromuscular disease, highlighting the need for the complete elucidation of the underlying molecular and pathophysiological mechanisms. Following a forward genetics approach with N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we identified a novel mouse model of autosomal recessive neuromuscular disease caused by a splice-site hypomorphic mutation in a novel gene of unknown function, DnaJC11. Recent findings have demonstrated that DNAJC11 protein co-immunoprecipitates with proteins of the mitochondrial contact site (MICOS) complex involved in the formation of mitochondrial cristae and cristae junctions. Homozygous mutant mice developed locomotion defects, muscle weakness, spasticity, limb tremor, leucopenia, thymic and splenic hypoplasia, general wasting and early lethality. Neuropathological analysis showed severe vacuolation of the motor neurons in the spinal cord, originating from dilatations of the endoplasmic reticulum and notably from mitochondria that had lost their proper inner membrane organization. The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog. The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization. Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells. Our findings provide the first mouse mutant for a putative MICOS protein and establish a link between DNAJC11 and neuromuscular diseases.

Show MeSH
Related in: MedlinePlus