Mitochondrial dysfunction in mut methylmalonic acidemia

Abstract

Methylmalonic acidemia is an autosomal recessive inborn error of metabolism caused by defective activity of methylmalonyl-CoA mutase (MUT) that exhibits multiorgan system pathology. To examine whether mitochondrial dysfunction is a feature of this organic acidemia, a background-modified Mut-knockout mouse model was constructed and used to examine mitochondrial ultrastructure and respiratory chain function in the tissues that manifest pathology in humans. In parallel, the liver from a patient with mut methylmalonic acidemia was studied in a similar fashion. Megamitochondria formed early in life in the hepatocytes of the Mut(-/-) animals and progressively enlarged. Liver extracts prepared from the mutants at multiple time points displayed respiratory chain dysfunction, with diminished cytochrome c oxidase activity and reduced intracellular glutathione compared to control littermates. Over time, the exocrine pancreas and proximal tubules of the kidney also exhibited megamitochondria, and older mutant mice eventually developed tubulointerstitial renal disease. The patient liver displayed similar morphological and enzymatic findings as observed in the murine tissues. These murine and human studies establish that megamitochondria formation with respiratory chain dysfunction occur in a tissue-specific fashion in methylmalonic acidemia and suggest treatment approaches based on improving mitochondrial function and ameliorating the effects of oxidative stress.

Figure 1.

a–d) Electron microscopy of liver samples from a control animal (a) and Mut^−/− knockout mice sacrificed on day 1 (b), day 7 (c), or day 32 (d). Many cellular elements can be seen in control mouse liver (a), including golgi network, endoplasmic reticulum, lipid droplets, and mitochondria. On day 1, mutant liver shows enlarged mitochondria and significantly increased lipid droplets (b). Changes progress, and by day 7 (c), mitochondria are much larger and pale, with diminished cristae. Lipid is present but is much less prominent than on day 1. By day 32 (d), mitochondria still retain a characteristic double membrane but are enormous, and cristae are barely visible at the periphery. e, f) Electron microscopy of kidney samples from a Mut^+/+ control animal (e) and Mut^−/− knockout littermate sacrificed on day 7 (f). Both pictures are from proximal tubule. Microvilli near brush border are present. Mitochondria in mutant are enlarged and have disorganized cristae. g, h) Electron microscopy of pancreas samples from a Mut^+/+ control animal (g) and Mut^−/− knockout littermate sacrificed on day 76 (h). Both pictures are from exocrine pancreas. Mitochondria in mutant are enlarged and display a pale matrix. Number of zymogen granules is less in the mutant than in the control, and endoplasmic reticulum is disorganized. C, cristae; ER, endoplasmic reticulum; LD, lipid droplets; MT, mitochondria; MV, microvilli; Z, zymogen. All views ×11,000. Scale bars =1 μm.

Figure 2.

Histology (hematoxylin and eosin stain) of kidney from a Mut^+/− control animal (a) and a [(C57BL/6×129 Sv/Ev) × FVB/N] Mut^−/− knockout littermate (b) sacrificed at 13 mo of age. In mutant (b), tubulointerstitial inflammation can be appreciated near arrow, and kidney outline is distorted. Some glomeruli (indicated by asterisk) are normal in mutant.

Figure 3.

a) Respiratory chain activity in murine liver extracts. Eight mutants from 4 different litters ranging from 10 to 30 days were studied with 9 age- and diet-matched littermates. Error bars surround the 95% confidence interval. Specific enzyme activities were measured as described in text and normalized to controls. Mut^−/− knockout mice show diminished activities of complex III and complex IV and increased citrate synthase. *P < 0.02; **P < 0.01; ***P < 0.0001. b) GSH and GSSG concentrations in liver extracts in mutant compared to heterozygote controls. *P < 0.01.

Figure 4.

a) Electron microscopy of a mut methylmalonic acidemia patient liver (×10,000). Lipid droplets (LD) and mitochondria (MT) are enlarged, have a pale matrix, and some have lamellar inclusions. b) Respiratory chain activity in a mut methylmalonic acidemia patient liver extract compared to controls (n=5). Specific enzyme activities were measured as described in text and normalized to controls. The mut methylmalonic acidemia patient liver shows diminished activities of complex IV and possibly complex II + III compared to controls.