Figure 1.
Clinical characterization of the spastic phenotype.
(A) Body weight curves of spastic (spc/spc) mice and sex matched control (+/+ or spc/+) littermates (n = 21 per group). (B) Kaplan–Meier survival curve of spc/spc mice and control (+/+ or spc/+) littermates (n = 38 per group). (C) Grip strength measurements of spc/spc mice and control (+/+ or spc/+), sex matched, littermates. Grip strength values were normalized with body weight to account for the reduced body size of spc/spc mice (n = 16). (D) Abnormal hind limb posture of spc/spc mice. (E) Loss of hind limb extension reflex when spc/spc mice are suspended by the tail. Mice shown are females and 3 weeks old littermates.
Figure 2.
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.
Figure 3.
Lymphoid and blood abnormalities in spc mice.
(A) Representative thymi dissected from spc/spc and WT (+/+) littermate mice at 4 weeks of age. Scalebar, 5mm. (B) Representative H/E stained thymic sections from spc/spc and +/+ littermate mice (n = 4). Scalebar, 400 µm. (C) Total thymus cellularity in spc/spc mice and control littermates, (n = 14 per group). (D) Percentage of thymic subpopulations in spc/spc mice and control littermates as determined by flow cytometry after staining of thymocytes with antibodies against CD4 and CD8. Data represent means ± SE from four independent experiments, (n = 13 per group). (E) Percentages of CD4-CD8- double negative (DN) subpopulations as determined by flow cytometry after staining of thymocytes with antibodies against CD25 and CD44, in spc/spc and control littermates, (n = 6 per group). (F) Representative spleens dissected from spc/spc and +/+ littermate mice at 4 weeks of age. Scalebar, 5 mm. (G) Total spleen cellularity of spc/spc mice and control littermates, (n = 14 per group). (H) Percentage of splenic subpopulations in spc/spc mice and control littermates as determined by flow cytometry using antibodies against CD4, CD8, B220 (B cells), Gr1 and CD11b (Myeloid). Data represent means ± SE from four independent experiments, (n = 10 per group). (I) Peripheral blood counts of spc/spc mice and control littermates, (n = 7 per group). Controls presented in bar graphs are healthy littermates (+/+ and +/spc).
Table 1.
Total Blood Counts in spc/spc mice.
Table 2.
Clinical chemistry in spc/spc mice.
Figure 4.
Mapping, identification and representation of the spc mutation.
(A) Through genome-wide linkage analysis the causal mutation was mapped on distal chromosome 4. (B) Representative RT-PCR analysis on cDNAs from the indicated tissues of spc/spc and WT (+/+) littermate mice using a primer pair specific for the 3′- terminal coding region of DnaJC11 transcript. Numbers indicate base pair lengths of DNA marker. Single and double asterisk indicate the 735 bp WT and 844 bp mutant PRC products that were sequenced, respectively. A Gluceraldehyde 3-phosphate dehydrogenase (GAPDH) primer pair was used as a control for cDNA synthesis. Same results were observed in three more mice. (C) DNA sequencing of the DnaJC11 gene in a WT control (+/+) and spc/spc littermate revealed an intronic T-to-A point mutation (asterisk). (D) Genomic organization of the DnaJC11 gene within exons 14-16 and indicated splicing sites for the WT (upper) and the spc (lower) transcript. The T-to-A mutation is indicated with a red asterisk and it generates a splice acceptor AG site which results in the incorporation of an 109 bp intronic region (exon X) into the mature transcript. The insertion introduces a premature stop codon (red underlining). Green highlighted is the physiological WT stop codon. Black line, splice donor sites; Grey lines, splice acceptor sites. (E) Primary structure of the 63 kDa muDNAJC11 protein. J denotes the J domain, regions in blue and red represent the DUF3395 domain and the predicted coiled coil region, respectively. Numbers denote amino acid residues. Red asterisk denotes the site of the mutation at the protein level and the red arrow the stretch of the protein that is predicted to be mutated due to the frameshift.
Figure 5.
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.
Figure 6.
Mitochondrial and submitochondrial localization of DNAJC11.
(A) Representative Western blot analysis on total protein RIPA extracts (T), on cytosolic (C) and mitochondrial (M) fractions of cerebrum and liver tissue from WT mice. The huDnaJC11 transgenic (Tg) samples served as positive controls. Prohibitin served as a mitochondrial specific marker and GAPDH as a cytoplasmic marker. (B) Fluorescence microscopy of HeLa cells transfected with a C-terminally FLAG-tagged huDNAJC11 cDNA of the 63 kDa isoform (green channel) and stained with the mitochondrial specific dye MitoTracker Orange (red channel). Scalebar, 100 µm. (C) Proteinase K protection assay on isolated mitochondria from HeLa cells. 50 µg of mitochondria were either subjected to swelling in the hypotonic buffer (SW, +) or were incubated in the isotonic buffer (SW, -) and then were treated with proteinase K (PK, +) or not (PK, -). A buffer containing 1% TritonX-100 (Tr) was used to solubilize all mitochondrial proteins and render them accessible to proteinase K. Samples were then analyzed by SDS-PAGE and Western blot with the indicated antibodies. Metaxin 1 is an outer membrane protein, mitofilin is an intermembrane space exposed inner membrane protein and Hsp60 is matrix localized. Numbers indicate molecular mass of protein marker in kDa. (D) Sodium carbonate extraction of isolated mitochondria from HeLa cells. Mitochondria were extracted under the two indicated pH conditions and the membranes were collected by ultracentrifugation. Pellet (P) and supernatant (S) were analyzed by SDS-PAGE and Western blot and probed with the indicated antibodies. Tim23 is an integral inner membrane protein and ICDH is a soluble matrix protein. Arrows indicate the 63 kDa isoform and the putative ∼57 kDa isoform. Tim23, Translocase of inner mitochondrial membrane 23 homolog; ICDH, isocitrate dehydrogenase.
Figure 7.
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.