Mice with mitochondrial complex I deficiency develop a fatal encephalomyopathy

Abstract

To study effects of mitochondrial complex I (CI, NADH:ubiquinone oxidoreductase) deficiency, we inactivated the Ndufs4 gene, which encodes an 18 kDa subunit of the 45-protein CI complex. Although small, Ndufs4 knockout (KO) mice appeared healthy until approximately 5 weeks of age, when ataxic signs began, progressing to death at approximately 7 weeks. KO mice manifested encephalomyopathy including a retarded growth rate, lethargy, loss of motor skill, blindness, and elevated serum lactate. CI activity in submitochondrial particles from KO mice was undetectable by spectrophotometric assays. However, CI-driven oxygen consumption by intact tissue was about half that of controls. Native gel electrophoresis revealed reduced levels of intact CI. These data suggest that CI fails to assemble properly or is unstable without NDUFS4. KO muscle has normal morphology but low NADH dehydrogenase activity and subsarcolemmal aggregates of mitochondria. Nonetheless, total oxygen consumption and muscle ATP and phosphocreatine concentrations measured in vivo were within normal parameters.

Figure 1. Phenotypes of Ndufs4 KO Mice

(A) Growth curves of WT, HET and KO female mice. KO mice weigh significantly less than either HET or WT. n =4 to 56, depending on day and genotype. Values given as mean ± s.d. p < 0.05 for all time points beyond P15, Student’s two-tailed t-test. (B) Locomotor activity of young (P30) KO mice become hypoactive. *, a significant difference (p =0.0001, Student’s two-tailed t-test) was obtained between old CT mice and old KO mice. Values given as mean ± s.e.m. (C) Oxygen consumption (liter/kg lean mass/hr) by KO mice (P26 to P37) during 72 hr in metabolic chambers was the same as controls during daytime, nighttime and fasting. n =9 (WT), 7 (KO), p =0.5 and greater for diurnal, fasting and nocturnal measurements. Values given as mean ± s.e.m. (D) Body temperature of KO mice older than P30 was ~2°C lower than controls. n =3–9, depending on day and genotype. p <0.001 for all days beyond P30, Student’s two-tailed t-test. Values given as mean ± s.e.m. (E) Electroretinograms indicate that KO mice (P21) lack the B-wave, indicating failure of neurotransmission from photoreceptors to bipolar cells. Superimposed traces from 4 WT and 2 KO mice. (F) Startle responses (arbitrary units) of KO mice were enhanced relative to WT mice until P36. n =14 (WT Inset, Repeated measurements of older KO mice (2 examples shown) reveal progressive loss of startle response, but with variable age of onset; SR (% max) is the percent of maximum startle response. (G) Rotarod performance (latency to fall) of young (20 (CT), n =4 (KO). Values given as mean ± s.e.m. *, p <0.0003 for all ages beyond P21, Student’s two-tailed t-test.

Figure 2. Respiratory Activities

(A) SMPs derived from KO liver had no CI activity, whereas CIII and CIV activities were not significantly different. CII activity was significantly higher. Data were normalized to citrate synthase activity (Δ Abs/min of respiratory complex activity : Δ Abs/min citrate synthase activity); hence they have no units. Assays in triplicate, n =12 for CI, CII, CIV; n =4 CIII. *, p =0.003; **, p =0.0001, Mann-Whitney test. Similar results were obtained with SMPs from brain and MEFs. Values given as mean ± s.e.m. (B) Rates of O₂ consumption by liver CI from WT and KO mice (P35) were measured. Arrows indicate addition of: (1) tissue, (2) ADP, (3) malate/pyruvate, and (4) rotenone. There was less O₂ consumption by KO tissue in the presence of malate/pyruvate (CI) relative to WT, and it stopped with addition of rotenone. (C) The ratio O₂ consumption (oxygen/mg tissue/min) by liver from KO versus WT mice. Complex I activity of KO tissue is less than half that of WT, whereas CII and CIV activities do not significantly differ, n =5. Values given as mean ± s.e.m. *, p =0.0001, Student’s two-tailed t-test. Similar results were obtained from brain and muscle. (D) Blue Native Gel Electrophoresis (BNGE) of CI from brain and liver mitochondria. Proteins representing the three major CI subcomplexes were examined by Western blot; in each case, KO mitochondria had less intact complex I. (E) SDS PAGE of proteins representing the three major subcomplexes of respiratory complex I. Equivalent amounts of mitochondrial protein were loaded, MnSOD was used as internal standard. The samples were the same as used for the BNGE experiment (Fig. 2D).

Figure 3. ATP Turnover Reactions During Ischemia Reperfusion in Hindlimb Muscles in vivo

(A) Representative data illustrating PCr and P_i dynamics during ischemia and reperfusion. Resting ATPase (equivalent to ATP demand) is equal to the initial rate of PCr breakdown during ischemia (black regression line). The phosphorylation capacity is calculated from the recovery of PCr following ischemia (Bliel et al., 1993). (B) Resting mitochondrial ATP production (equal to resting ATP demand) is not different in WT (black bars) and KO (open bars) mice. Values given as mean ± s.e.m. (c) The phosphorylation capacity is not significantly different in the skeletal muscle of the WT and KO mice, P =0.12; n =6 for both WT and KO groups. Values given as mean ± s.e.m.

Figure 4. Histological Comparisons of Muscle from KO and WT Mice

(A) Electron microscopic images of the subsarcolemmal layer of soleus muscle (P35). Larger subsarcolemmal clusters of mitochondria (6 or more mitochondria deep) were observed in 50% of soleus muscle fibers from KO mice versus no fibers with clusters greater than 5 mitochondria deep in WT soleus. (B) Light microscopy of muscle from a WT and KO mouse (age P23, soleus; a highly oxidative muscle). Glycolytic (yellow arrows) and oxidative fibers (red arrows) are indicated. H & E, Haemotoxylin and eosin staining. Gomori trichrome staining; there are no “ragged-red” fibers. Myofibrillar ATPase staining reveals fiber types. Quantitative analysis of slides of 4 mice of each genotype revealed no significant difference in fraction of glycolytic fibers. SDH assay stain intensity often appears slightly darker for KO mice, especially in oxidative muscle fibers identified by ATPase staining. Quantitative analysis of slides of 5 mice of each genotype revealed no significant difference. NADH oxidase activity of soleus muscle is lower in KO muscle fibers, primarily in the slow oxidative type (type I, black arrows); white arrows point to glycolytic (type IIB) fibers. COX, Cytochrome oxidase staining of KO mice is comparable to WT.