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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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Complete rescue of the DnaJC11spc/spc phenotype through expression of the human DnaJC11 gene.(A) Schematic representation of the human BAC clone fragment that was used for the generation of TghuDnaJC11 mice. Genes and their orientation are indicated as well as NotI sites that were used for digestion. Horizontal line and number below represent the fragment length. (B) Copy number determination, by qPCR, of three transgenic lines, TgF843, TgF867, and TgF869 (n = 5-9 per group) using a primer pair common for both mouse and human DnaJC11 genes. WT mice were considered to carry 2 copies of DnaJC11. (C) Body weight and (D) grip strength (normalized to body weight) curves for the indicated genotypes. All mice used were sex matched littermates, (n = 8). (E) Rescue of the thymic hypoplasia shown as total thymic cellularity, (n = 3). (F) Restoration of thymic subpopulations distribution in rescued mice (n = 3) as determined by flow cytometry after staining with antibodies against CD4 and CD8. Statistical analysis between controls and rescued (Tg/DnaJC11spc/spc) mice is indicated. DP, double positive; DN, Double Negative. (G) Restoration of splenic subpopulations distribution in rescued mice. B cells and myeloid cells were defined as the ones positive for markers B220 and CD11b respectively, (n = 3). (H) Restoration of the leucopenia phenotype and the increased red blood cell phenotype in rescued mice (n = 3). Data represent means ± SE.
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pone-0104237-g005: Complete rescue of the DnaJC11spc/spc phenotype through expression of the human DnaJC11 gene.(A) Schematic representation of the human BAC clone fragment that was used for the generation of TghuDnaJC11 mice. Genes and their orientation are indicated as well as NotI sites that were used for digestion. Horizontal line and number below represent the fragment length. (B) Copy number determination, by qPCR, of three transgenic lines, TgF843, TgF867, and TgF869 (n = 5-9 per group) using a primer pair common for both mouse and human DnaJC11 genes. WT mice were considered to carry 2 copies of DnaJC11. (C) Body weight and (D) grip strength (normalized to body weight) curves for the indicated genotypes. All mice used were sex matched littermates, (n = 8). (E) Rescue of the thymic hypoplasia shown as total thymic cellularity, (n = 3). (F) Restoration of thymic subpopulations distribution in rescued mice (n = 3) as determined by flow cytometry after staining with antibodies against CD4 and CD8. Statistical analysis between controls and rescued (Tg/DnaJC11spc/spc) mice is indicated. DP, double positive; DN, Double Negative. (G) Restoration of splenic subpopulations distribution in rescued mice. B cells and myeloid cells were defined as the ones positive for markers B220 and CD11b respectively, (n = 3). (H) Restoration of the leucopenia phenotype and the increased red blood cell phenotype in rescued mice (n = 3). Data represent means ± SE.

Mentions: To confirm that the intronic point mutation identified in the muDnaJC11 gene was indeed causal of the phenotype developed in the spc/spc (DnaJC11spc/spc) mice, genetic rescue experiments were performed by generating transgenic mice (TghuDnaJC11) that carry the whole genomic region of human DnaJC11 (huDnaJC11) and crossing them into the DnaJC11spc/spc background. As the human and mouse 63 kDa DNAJC11 proteins share 97% identity and 99% homology, they are expected to perform redundant functions. An approximately 120 kb genomic fragment containing the whole huDnaJC11 gene was isolated from a bacterial artificial chromosome (BAC) clone (Figure 5A) and was used for pronuclear microinjections. The BAC fragment that was used contained also part of the Thap3 gene but all the 5′-region of this gene up to the base encoding the first 89 amino acids of the respective protein was excluded. Thus, no protein from this gene is expected to be produced. Upon microinjections, seven transgenic founders were obtained, all of which were fertile and appeared healthy, exhibiting no obvious neurological symptoms. Out of the seven founders, three (TgF843, TgF867 and TgF869) were chosen to establish transgenic lines in order to use them in rescue experiments. Copy number determination by quantitative real-time PCR (qPCR) using a primer pair common for the human and mouse DnaJC11 showed that all three transgenic lines carried one or two transgene copies (Figure 5B). qPCR for TgF869 line, which carried 2 copies of the transgene, verified a 2 to 6 fold increase of DnaJC11 transcript levels depending on the tissue (Figure S9A). huDNAJC11 overexpression was also verified in Western blot analyses for many tissues (Figure S9B). Crossing each of the three transgenic lines into the DnaJC11spc/spc background completely rescued the premature lethality, growth retardation and the reduced grip strength displayed by the DnaJC11spc/spc mice (Figure 5C-D). No neurological symptoms manifested in these rescued lines within a period of one year. The TgF869 line was further investigated in rescue experiments. Indeed, in TgF869/DnaJC11spc/spc mice the motor neuron vacuolation phenotype and abnormal cristae structures (Figure S10A-B), the thymic and splenic hypoplasia, the thymic and splenic subpopulations phenotype, and the leucopenia were completely rescued (Figure 5E-H). These data genetically confirm the causal role of the DnaJC11 mutation in the DnaJC11spc/spc phenotype and that the mouse and human genes have redundant functions.


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)

Complete rescue of the DnaJC11spc/spc phenotype through expression of the human DnaJC11 gene.(A) Schematic representation of the human BAC clone fragment that was used for the generation of TghuDnaJC11 mice. Genes and their orientation are indicated as well as NotI sites that were used for digestion. Horizontal line and number below represent the fragment length. (B) Copy number determination, by qPCR, of three transgenic lines, TgF843, TgF867, and TgF869 (n = 5-9 per group) using a primer pair common for both mouse and human DnaJC11 genes. WT mice were considered to carry 2 copies of DnaJC11. (C) Body weight and (D) grip strength (normalized to body weight) curves for the indicated genotypes. All mice used were sex matched littermates, (n = 8). (E) Rescue of the thymic hypoplasia shown as total thymic cellularity, (n = 3). (F) Restoration of thymic subpopulations distribution in rescued mice (n = 3) as determined by flow cytometry after staining with antibodies against CD4 and CD8. Statistical analysis between controls and rescued (Tg/DnaJC11spc/spc) mice is indicated. DP, double positive; DN, Double Negative. (G) Restoration of splenic subpopulations distribution in rescued mice. B cells and myeloid cells were defined as the ones positive for markers B220 and CD11b respectively, (n = 3). (H) Restoration of the leucopenia phenotype and the increased red blood cell phenotype in rescued mice (n = 3). Data represent means ± SE.
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pone-0104237-g005: Complete rescue of the DnaJC11spc/spc phenotype through expression of the human DnaJC11 gene.(A) Schematic representation of the human BAC clone fragment that was used for the generation of TghuDnaJC11 mice. Genes and their orientation are indicated as well as NotI sites that were used for digestion. Horizontal line and number below represent the fragment length. (B) Copy number determination, by qPCR, of three transgenic lines, TgF843, TgF867, and TgF869 (n = 5-9 per group) using a primer pair common for both mouse and human DnaJC11 genes. WT mice were considered to carry 2 copies of DnaJC11. (C) Body weight and (D) grip strength (normalized to body weight) curves for the indicated genotypes. All mice used were sex matched littermates, (n = 8). (E) Rescue of the thymic hypoplasia shown as total thymic cellularity, (n = 3). (F) Restoration of thymic subpopulations distribution in rescued mice (n = 3) as determined by flow cytometry after staining with antibodies against CD4 and CD8. Statistical analysis between controls and rescued (Tg/DnaJC11spc/spc) mice is indicated. DP, double positive; DN, Double Negative. (G) Restoration of splenic subpopulations distribution in rescued mice. B cells and myeloid cells were defined as the ones positive for markers B220 and CD11b respectively, (n = 3). (H) Restoration of the leucopenia phenotype and the increased red blood cell phenotype in rescued mice (n = 3). Data represent means ± SE.
Mentions: To confirm that the intronic point mutation identified in the muDnaJC11 gene was indeed causal of the phenotype developed in the spc/spc (DnaJC11spc/spc) mice, genetic rescue experiments were performed by generating transgenic mice (TghuDnaJC11) that carry the whole genomic region of human DnaJC11 (huDnaJC11) and crossing them into the DnaJC11spc/spc background. As the human and mouse 63 kDa DNAJC11 proteins share 97% identity and 99% homology, they are expected to perform redundant functions. An approximately 120 kb genomic fragment containing the whole huDnaJC11 gene was isolated from a bacterial artificial chromosome (BAC) clone (Figure 5A) and was used for pronuclear microinjections. The BAC fragment that was used contained also part of the Thap3 gene but all the 5′-region of this gene up to the base encoding the first 89 amino acids of the respective protein was excluded. Thus, no protein from this gene is expected to be produced. Upon microinjections, seven transgenic founders were obtained, all of which were fertile and appeared healthy, exhibiting no obvious neurological symptoms. Out of the seven founders, three (TgF843, TgF867 and TgF869) were chosen to establish transgenic lines in order to use them in rescue experiments. Copy number determination by quantitative real-time PCR (qPCR) using a primer pair common for the human and mouse DnaJC11 showed that all three transgenic lines carried one or two transgene copies (Figure 5B). qPCR for TgF869 line, which carried 2 copies of the transgene, verified a 2 to 6 fold increase of DnaJC11 transcript levels depending on the tissue (Figure S9A). huDNAJC11 overexpression was also verified in Western blot analyses for many tissues (Figure S9B). Crossing each of the three transgenic lines into the DnaJC11spc/spc background completely rescued the premature lethality, growth retardation and the reduced grip strength displayed by the DnaJC11spc/spc mice (Figure 5C-D). No neurological symptoms manifested in these rescued lines within a period of one year. The TgF869 line was further investigated in rescue experiments. Indeed, in TgF869/DnaJC11spc/spc mice the motor neuron vacuolation phenotype and abnormal cristae structures (Figure S10A-B), the thymic and splenic hypoplasia, the thymic and splenic subpopulations phenotype, and the leucopenia were completely rescued (Figure 5E-H). These data genetically confirm the causal role of the DnaJC11 mutation in the DnaJC11spc/spc phenotype and that the mouse and human genes have redundant functions.

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