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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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Neuronal vacuolation and abnormal mitochondria in the CNS of spc/spc mice.(A) Representative toluidine blue stained resin sections of motor neurons in the lumbar segment of the spinal cord (a,b,d,e) and neurons in the medulla (c,f) from WT littermates (a-c) and spc/spc mice (d-f). Lumbar, (n = 5 in three different experiments), medulla (n = 3 in two different experiments). Scalebar, 20 µm. Arrows indicate vacuoles and asterisks glial cells. (B) Representative electron micrographs of motor neuron cell bodies (a-g) and terminal axons (h-i) in the region of the ventral horn of the spinal cord from spc/spc (a-f and h) and WT control mice (g and i). (a), low power magnification of a vacuolated motor neuron. (b-g), high power magnification in the cell bodies of motor neurons. (b-e) mitochondria with disrupted cristae (arrows) or others with abnormally stacked or concentric membranes (asterisks) can be identified. Arrows in c indicate double membrane bound vacuoles completely electron transparent, devoid of cristae. (f), single membrane bound vacuoles (arrows) possibly originating from the ER. (g), a normal WT mitochondrion (arrow). (h-i), synaptic mitochondria (arrows) in terminal axons identified by the presence of prosynaptic vesicles. WT (n = 2), spc/spc (n = 4). Scalebars: a, 2 µm; b, d-e, 100 nm, c, f-i, 200 nm.
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pone-0104237-g002: Neuronal vacuolation and abnormal mitochondria in the CNS of spc/spc mice.(A) Representative toluidine blue stained resin sections of motor neurons in the lumbar segment of the spinal cord (a,b,d,e) and neurons in the medulla (c,f) from WT littermates (a-c) and spc/spc mice (d-f). Lumbar, (n = 5 in three different experiments), medulla (n = 3 in two different experiments). Scalebar, 20 µm. Arrows indicate vacuoles and asterisks glial cells. (B) Representative electron micrographs of motor neuron cell bodies (a-g) and terminal axons (h-i) in the region of the ventral horn of the spinal cord from spc/spc (a-f and h) and WT control mice (g and i). (a), low power magnification of a vacuolated motor neuron. (b-g), high power magnification in the cell bodies of motor neurons. (b-e) mitochondria with disrupted cristae (arrows) or others with abnormally stacked or concentric membranes (asterisks) can be identified. Arrows in c indicate double membrane bound vacuoles completely electron transparent, devoid of cristae. (f), single membrane bound vacuoles (arrows) possibly originating from the ER. (g), a normal WT mitochondrion (arrow). (h-i), synaptic mitochondria (arrows) in terminal axons identified by the presence of prosynaptic vesicles. WT (n = 2), spc/spc (n = 4). Scalebars: a, 2 µm; b, d-e, 100 nm, c, f-i, 200 nm.

Mentions: The motor defects observed in spc/spc mice prompted us to investigate for motor neuron pathology. Because of the severity of the symptoms in the hind limbs, we firstly examined cross sections of the lumbar segment of the spinal cord from 4 weeks old spc/spc mice. Motor neurons in the anterior horn of WT littermates were properly maintained with obvious Nissl substance, nuclei and nucleoli (Figure 2Aa-b), whereas these cells in the spc/spc mice appeared completely vacuolated (Figure 2Ad-e). These vacuoles were mainly confined in the cell bodies of motor neurons but also extended to dendrites (Figure 2Ae). They were roughly circular and of various sizes and appeared either opaque (Figure 2Ad) or transparent (Figure 2Ae), usually filling the whole body of the neuron. No such vacuoles were observed in the neuropil, the white matter, the dorsal horn neurons or in glial cells of the spinal cord. Nuclei and nucleoli of motor neurons appeared normal (Figure 2A). The same analysis was performed for various regions of the brain. Again, cell structure in the WT mice was well maintained and no vacuolation was observed (Figure 2Ac). In the spc/spc mice though, vacuolation was present mainly in the medulla (Figure 2Af), which was affected to a lesser extent compared to the spinal cord with regard to the number of affected neurons and the number of vacuoles in the affected neurons.


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)

Neuronal vacuolation and abnormal mitochondria in the CNS of spc/spc mice.(A) Representative toluidine blue stained resin sections of motor neurons in the lumbar segment of the spinal cord (a,b,d,e) and neurons in the medulla (c,f) from WT littermates (a-c) and spc/spc mice (d-f). Lumbar, (n = 5 in three different experiments), medulla (n = 3 in two different experiments). Scalebar, 20 µm. Arrows indicate vacuoles and asterisks glial cells. (B) Representative electron micrographs of motor neuron cell bodies (a-g) and terminal axons (h-i) in the region of the ventral horn of the spinal cord from spc/spc (a-f and h) and WT control mice (g and i). (a), low power magnification of a vacuolated motor neuron. (b-g), high power magnification in the cell bodies of motor neurons. (b-e) mitochondria with disrupted cristae (arrows) or others with abnormally stacked or concentric membranes (asterisks) can be identified. Arrows in c indicate double membrane bound vacuoles completely electron transparent, devoid of cristae. (f), single membrane bound vacuoles (arrows) possibly originating from the ER. (g), a normal WT mitochondrion (arrow). (h-i), synaptic mitochondria (arrows) in terminal axons identified by the presence of prosynaptic vesicles. WT (n = 2), spc/spc (n = 4). Scalebars: a, 2 µm; b, d-e, 100 nm, c, f-i, 200 nm.
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pone-0104237-g002: Neuronal vacuolation and abnormal mitochondria in the CNS of spc/spc mice.(A) Representative toluidine blue stained resin sections of motor neurons in the lumbar segment of the spinal cord (a,b,d,e) and neurons in the medulla (c,f) from WT littermates (a-c) and spc/spc mice (d-f). Lumbar, (n = 5 in three different experiments), medulla (n = 3 in two different experiments). Scalebar, 20 µm. Arrows indicate vacuoles and asterisks glial cells. (B) Representative electron micrographs of motor neuron cell bodies (a-g) and terminal axons (h-i) in the region of the ventral horn of the spinal cord from spc/spc (a-f and h) and WT control mice (g and i). (a), low power magnification of a vacuolated motor neuron. (b-g), high power magnification in the cell bodies of motor neurons. (b-e) mitochondria with disrupted cristae (arrows) or others with abnormally stacked or concentric membranes (asterisks) can be identified. Arrows in c indicate double membrane bound vacuoles completely electron transparent, devoid of cristae. (f), single membrane bound vacuoles (arrows) possibly originating from the ER. (g), a normal WT mitochondrion (arrow). (h-i), synaptic mitochondria (arrows) in terminal axons identified by the presence of prosynaptic vesicles. WT (n = 2), spc/spc (n = 4). Scalebars: a, 2 µm; b, d-e, 100 nm, c, f-i, 200 nm.
Mentions: The motor defects observed in spc/spc mice prompted us to investigate for motor neuron pathology. Because of the severity of the symptoms in the hind limbs, we firstly examined cross sections of the lumbar segment of the spinal cord from 4 weeks old spc/spc mice. Motor neurons in the anterior horn of WT littermates were properly maintained with obvious Nissl substance, nuclei and nucleoli (Figure 2Aa-b), whereas these cells in the spc/spc mice appeared completely vacuolated (Figure 2Ad-e). These vacuoles were mainly confined in the cell bodies of motor neurons but also extended to dendrites (Figure 2Ae). They were roughly circular and of various sizes and appeared either opaque (Figure 2Ad) or transparent (Figure 2Ae), usually filling the whole body of the neuron. No such vacuoles were observed in the neuropil, the white matter, the dorsal horn neurons or in glial cells of the spinal cord. Nuclei and nucleoli of motor neurons appeared normal (Figure 2A). The same analysis was performed for various regions of the brain. Again, cell structure in the WT mice was well maintained and no vacuolation was observed (Figure 2Ac). In the spc/spc mice though, vacuolation was present mainly in the medulla (Figure 2Af), which was affected to a lesser extent compared to the spinal cord with regard to the number of affected neurons and the number of vacuoles in the affected neurons.

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