Cisd2 deficiency drives premature aging and causes mitochondria-mediated defects in mice

Abstract

CISD2, the causative gene for Wolfram syndrome 2 (WFS2), is a previously uncharacterized novel gene. Significantly, the CISD2 gene is located on human chromosome 4q, where a genetic component for longevity maps. Here we show for the first time that CISD2 is involved in mammalian life-span control. Cisd2 deficiency in mice causes mitochondrial breakdown and dysfunction accompanied by autophagic cell death, and these events precede the two earliest manifestations of nerve and muscle degeneration; together, they lead to a panel of phenotypic features suggestive of premature aging. Our study also reveals that Cisd2 is primarily localized in the mitochondria and that mitochondrial degeneration appears to have a direct phenotypic consequence that triggers the accelerated aging process in Cisd2 knockout mice; furthermore, mitochondrial degeneration exacerbates with age, and the autophagy increases in parallel to the development of the premature aging phenotype. Additionally, our Cisd2 knockout mouse work provides strong evidence supporting an earlier clinical hypothesis that WFS is in part a mitochondria-mediated disorder; specifically, we propose that mutation of CISD2 causes the mitochondria-mediated disorder WFS2 in humans. Thus, this mutant mouse provides an animal model for mechanistic investigation of Cisd2 protein function and help with a pathophysiological understanding of WFS2.

Figures

Figure 1.

The decreased body weight, the shortened life span, and the ocular and cutaneous symptoms of aging in Cisd2−/− mice. (A) Growth curves of the different genotypes. (B) Decreased survival rate of the Cisd2−/− mice. (C) The prominent eyes and protruding ears of the Cisd2−/− mice. (D) The Cisd2−/− mice go blind at ~6 mo old. (E) The opacity of cornea was analyzed by histological examination. Masson's trichrome staining indicated debris deposition in the scar tissue outside the cornea. (F) Early depigmentation and gray hair are present on top of the head and on the shoulders. The representative photo was taken from a 12-mo-old Cisd2−/− female. (G) Hair follicle atrophy in the Cisd2−/− mice was demonstrated by Masson's trichrome staining. (H) A decreased density of hair follicles containing hair in the Cisd2−/− mice was detected compared with wild-type skin. (I,J) Histological analyses of the skin from 12-mo-old wild-type and Cisd2−/− mice, respectively. The Cisd2−/− skin exhibits a phenotype involving a hyperplastic epidermis, hair follicle atrophy, a decrease in subcutaneous fat and muscle, and an increased thickness of the dermis layer. (K) Quantification of the subcutaneous muscle tissue, adipose tissue, and dermis for the histological sections of the wild-type and Cisd2−/− skins. (*) P < 0.05 was considered statistically significant.

Figure 2.

Abnormalities of skeleton and muscle and decrease of thoracic volume in the Cisd2−/− mice. (A) Micro-CT imaging of the femur trabecula at 12 wk old. Thinner trabecular thickness was observed in the Cisd2−/− mouse. (B) Osteopenia was analyzed by DEXA. A decrease in femur density was detected with the 8-wk-old Cisd2−/− mice. At 24 wk old, a decrease in femur density also was detected with the Cisd2+/− mice and, at 24 wk old, the phenotype had become more severe in the Cisd2−/− mice. (C) Radiographs of a wild-type mouse and a Cisd2−/− mouse at 16 wk old. The Cisd2−/− mouse displays a lordokyphosis (curvature of the spinal column) phenotype. (D) Micro-CT scanning allowed three-dimensional reconstruction of the thoracic and spinal column. (E) Lordokyphosis was evident and had led to a decrease in thoracic volume in the Cisd2−/− mice compared with their wild-type littermates. (F,G) H&E staining of transverse sections of skeletal muscle of 4-wk-old and 28-mo-old wild-type mice. (H,I) Muscle degeneration of 4-wk-old and 8-wk-old Cisd2−/− mice was examined by H&E staining of transverse sections of the skeletal muscle. Black arrows indicate degenerated transverse fibers that are present in the Cisd2−/− and also in spontaneously aged mice. The blue arrow indicates an angular fiber, which is an indicator of muscle atrophy caused by neuron degeneration. (J) Quantification of the degenerating fibers in the skeletal muscles. (*) P < 0.05; (**) P < 0.005.

Figure 3.

Mitochondrial degeneration and autophagy induction in the muscles and neurons of the Cisd2−/− mice. (A) Wild-type mitochondria in the brain (hippocampus). (B) A Cisd2−/− mitochondrion in the brain (hippocampus). Note that the outer mitochondrial membrane has broken down (arrowhead), while the inner cristae appear to be intact. (C) Cisd2−/− mitochondria in sciatic nerve. One mitochondrion (arrowhead) has a destroyed OM, but with cristae still visible; the other mitochondrion (arrow) has destroyed OMs and IMs. (D) Wild-type mitochondria in cardiac muscle. (E) Cisd2−/− mitochondria in cardiac muscle. This micrograph shows one mitochondrion (arrowhead) with a destroyed OM and two degenerated mitochondria consisting of debris (arrows). (F) A cluster of autophagic vacuoles and abnormal mitochondria was observed between the myofibrils of Cisd2−/− skeletal muscle (white arrows). (G) A wild-type myelinated axon of the sciatic nerve. (N) Nucleus of Schwann cell; (MS) myelin sheath. (H) A myelinated axon of sciatic nerve from a Cisd2−/− mouse. An ovoid with a disintegrating myelin sheath and a degenerating axonal component are shown. (I) Debris from an axon undergoing degeneration in the Cisd2−/− sciatic nerve. (J–L) Early or AVis enclosing mitochondria (arrows) and late or AVds were detected in the axonal component and cytoplasm of a Schwann cell from a 2-wk-old Cisd2−/− sciatic nerve. (M,N) Percentage of myelinated axons present in the sciatic nerves showing disintegration of their myelin sheaths and autophagic vacuoles, including AVi and AVd, in their axonal component. There were three mice for each group. (O) Western blotting to detected the presence of the proteins LC3-I and LC3-II. (P) Ratios of the LC3-II to LC3-I. There were three mice for each group. (*) P < 0.05; (**) P < 0.005. Mouse age in A–I is 4 wk old.

Figure 4.

Cisd2 is primarily localized in the outer mitochondrial membrane, and Cisd2 deficiency leads to mitochondrial dysfunction. (A) EGFP-tagged Cisd2 protein is directed to the mitochondria by an N-terminal signal sequence. The EGFP-Cisd2 proteins were expressed in NIH3T3 cells. EGFP-tagged full-length Cisd2 protein was colocalized with MitoTracker Red, whereas deletion of the N-terminal 58 amino acids completely abolished mitochondria localization. When the N-terminal 58-amino-acid sequence was fused to EGFP, this construct was able to redirect EGFP from a nuclear and cytoplasmic localization to the mitochondria. (B) Subcellular localization of the Cisd2 and Cisd1 proteins analyzed by Western blotting using protein extracts of the mitochondrial (Mito) and cytosolic (Cyto) fractions prepared from skeletal muscles of 12-wk-old mice. Polyclonal antibody (Ab) against Cisd2 protein (15 kDa) was generated; this antibody cross-reacts with Cisd1 protein (12 kDa). Antibodies against mitochondrial proteins Cisd1 and Hsp60 were used as controls. (C) Ten micrograms of each submitochondrial fraction prepared from the livers of 4-wk-old mice were analyzed by Western blot using antibodies against Cisd2 and known mitochondrial marker proteins. OM marker: (VDAC-1) voltage-dependent anion channel-1; IM marker: (ATP5B) complex V β subunit; matrix marker: (PDH) pyruvate dehydrogenase. (MP) Microplast (IM and matrix); (IMS) intermembrane space. (D) Impaired mitochondrial respiration in the skeletal muscle of 4-wk-old Cisd2−/− mice. Representative oxygraphs of the mitochondria after adding first glutamate-malate and then ADP into the closed chamber of the oxygen meter. (E) Respiratory activity was expressed as oxygen consumption rate (nanomoles of O2 per minute per milligram of mitochondria) in the resting state, for glutamate-malate supported respiration, and for ADP activated respiration. A significant decrease in oxygen consumption was detected in the Cisd2−/− mitochondrial samples (n = 4) compared with wild-type samples (n = 3). (F) The RCR (O2 consumption rate after ADP addition/O2 consumption rate after glutamate-malate addition) was significantly lower in the Cisd2−/− mitochondria. (G) Comparison of electron transport activities of the respiratory enzyme complexes of mitochondria prepared from the skeletal muscles of 4-wk-old Cisd2−/− (n = 4) and wild-type mice (n = 4). (NCCR activity) Measurement of NCCR activity, which represents complexes I–III; (SCCR activity) measurement of SCCR activity, which represents complexes II and III; (CCO activity) cCCO activity, which represents complex IV. (*) P < 0.05; (**) P < 0.005.

Figure 5.

Optic nerve degeneration and impaired glucose tolerance in Cisd2−/− mice. (A) A representative TEM micrograph showing a late or AVd detected in the axonal component of a myelinated axon of the optic nerve in 24-wk-old Cisd2−/− mice. The white arrow indicates a disintegrating myelinated axon. (B) Percentage of myelinated axons of the optic nerves containing autophagic vacuoles, including AVi and AVd, in the axonal component. There were three mice for each group; (wk) week. (C,D) Blood glucose levels and plasma insulin levels, respectively, before (0 min) and after the glucose load at the indicated time points. Oral glucose (1.5 g/kg body weight) tolerance tests were performed on 12-wk-old Cisd2−/− and wild-type mice, all of which had a C57BL/6 genetic background. Blood samples were collected to determine the mice's blood glucose levels and plasma insulin levels. (E) Insulin (0.75 U/kg body weight) tolerance tests were performed on 12-wk-old Cisd2−/− and wild-type mice. There were three mice in each group, and three independent measurements were carried out on each mouse. (*) P < 0.05; (**) P < 0.005. (F) IHC staining of insulin in the β cells of pancreatic islets using tissue sections prepared from 12-wk-old Cisd2−/− and wild-type mice.

Figure 6.

Summary of the aging-related phenotypes as a function of age in the Cisd2−/− mice. The timing of the onset of each phenotype approximates the average age of onset for that phenotype; (wk) week. The onset age for each mouse for each phenotype shows variation around the average onset age to a limited degree.