Mitochondrial Alterations Caused by Defective Peroxisomal Biogenesis in a Mouse Model for Zellweger Syndrome (PEX5 Knockout Mouse)

Materials for Morphological Investigations

All components for the preparation of Epon 812 were purchased from Fluka (Neu-Ulm, Germany). Paraffin (Paraplast plus) was from Sherwood Medical (St. Louis, MO). LR White, Unicryl, and osmium tetroxide were obtained from Polysciences (Heidelberg, Germany). Glutaraldehyde (25% stock solution), paraformaldehyde, PIPES, 3,3′-diaminobenzidine, cytochrome c, and di-sodium-cytidine-monophosphate were from Sigma (München, Germany). All other chemicals used for morphological experiments were obtained from Merck (Darmstadt, Germany).

Materials for Biochemical Investigations

Lactate dehydrogenase from hog muscle (complex V measurement), pyruvate kinase from rabbit muscle, 3-phosphoglycerate kinase from yeast, glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscle, β-hydroxybutyrate dehydrogenase grade II from Rhodobacter spheroides, lactate dehydrogenase from rabbit muscle (lactate and pyruvate measurements), glutamate-pyruvate transaminase from pig heart, NAD, and fat-free bovine serum albumin were purchased from Roche (Mannheim, Germany). Coenzyme Q1, decylubiquinone, cytochrome c from horse heart, NADH, n-dodecyl-β-d-maltoside, phosphoenolpyruvate, rotenone, antimycin A, oligomycin, glycerate-3-phosphate, lactate, pyruvate, and acetoacetate were from Sigma (Bornem, Belgium), ATP from Acros (Geel, Belgium), β-hydroxybutyrate from Serva (Heidelberg, Germany), PD-10 columns, Hybond-N+ nylon membranes and Protran nitrocellulose transfer membranes from Amersham Pharmacia Biotech Benelux (Roosendaal, The Netherlands), Dulbecco’s modified Eagle’s medium, fetal calf serum, l-glutamine, gentamicin, and Trizol reagent from Life Technologies (Merelbeke, Belgium), primers for polymerase chain reaction were obtained from Eurogentec (Seraing, Belgium).

Primary Antibodies

The monospecificity of the polyclonal rabbit anti-rat liver catalase antibody was characterized previously. 25 Mouse monoclonal antibodies against the following subunits of the mitochondrial respiratory chain complexes were purchased from Molecular Probes (Leiden, The Netherlands): complex I, anti-bovine NADH-ubiquinol oxidoreductase 39-kd subunit (clone 20C11-B11-B11); complex II, anti-bovine succinate-ubiquinol oxidoreductase 30-kd subunit (clone 21A11-AE7); complex III, anti-bovine ubiquinol-cytochrome c oxidoreductase core 1 subunit (clone 13G12AF12-BB11); complex IV, anti-human cytochrome c oxidase subunit 1 (clone D6-E1-A8); complex V, anti-bovine mitochondrial ATP synthase α subunit (clone 7H10-BD4).

Polyclonal rabbit anti-rat manganese-superoxide dismutase (MnSOD) antibodies were purchased from Research Diagnostics Inc. (Flanders, NJ). In addition, for immunohistochemical studies of MnSOD, we used a polyclonal rabbit anti-rat MnSOD antibody, which was a generous gift of Prof. K. Kato (Aichi Human Service Center, Aichi, Japan). 26

PEX5 Knockout Mice

Homozygous PEX5^−/− mice were obtained by breeding of the heterozygotes and identified by genotyping of tail DNA. They showed all characteristic metabolic changes and pathological alterations as described recently. 1 Pregnancy state of heterozygous mothers was calculated according to the appearance of the vaginal plug. Animals of embryonic day 18.5 (E18.5) or newborn mice (P0.5) were used for all investigations. The morning of the appearance of a vaginal plug was considered as E0.5. Pregnant mothers had access to food and water ad libitum and were exposed to a 12-hour light cycle.

Morphological Experiments

Biochemical Experiments

Mitochondria in Hepatocytes from PEX5^−/− Mice Proliferate and Show Severe Ultrastructural Abnormalities

As previously described, 1 regular peroxisomes were easily identified by histochemical staining for catalase with 3,3′diaminobenzidine in hepatocytes of control mice at gestational day 18.5 (E18.5) (Figure 1A) ▶ , but were absent in the PEX5 knockouts of the same age (Figure 1B) ▶ . In ∼5% of hepatocytes of PEX5-deficient mice peroxisomal membrane-remnant ghosts consisting of double-membraned loops measuring ∼100 nm across were observed that were labeled with an antibody to 70-kd peroxisomal membrane protein. 1 Similar structures were also observed in the adrenal glands but not in the kidney tubules (data not shown).

Strikingly, large aggregates of pleomorphic mitochondria were observed in many hepatocytes of PEX5-deficient mice, indicating focal proliferation of the mitochondrial compartment (Figure 1B) ▶ . Such mitochondrial aggregates were randomly distributed in the liver and were often found under the sinusoidal and the basolateral surface of hepatocytes (Figure 1B) ▶ . By electron microscopy different types of alterations of mitochondria were present, which were more pronounced in newborn (Figure 1; G to J ▶ ) as opposed to E18.5 PEX5^−/− mice (Figure 1; C to F ▶ ). These alterations involved all subcompartments of the mitochondria (outer membrane, inner membrane, and the matrix space). Alterations of the outer membrane consisted of 1) membrane whorls on the surface of mitochondria (Figure 1E) ▶ , 2) small blebs with electronlucent contents (Figure 1G) ▶ , or 3) protrusions of the inner membrane through focal interruptions of the outer membrane (Figure 1F) ▶ . In mitochondrial cristae, more obvious morphological changes were observed that consisted of 1) rarefication of cristae (Figure 1, B and J) ▶ , 2) arrangements into parallel stacks (Figure 1, B and F) ▶ , 3) curvilinear and circular forms (Figure 1; C and H to J ▶ ), 4) onion-like concentric rings (Figure 1, D and G) ▶ , 5) dilation with the formation of tubular cristae similar to those seen in steroid hormone-producing cells (Figure 1E) ▶ . In hepatocytes of newborn PEX5 knockout mice, we also observed ghost-like mitochondrial rest structures (Figure 1, H and I ▶ ; small arrows), swollen mitochondria with strong rarefication of cristae, and reduced electron density of their matrix (Figure 1, I and J ▶ ; asterisks). Mitochondrial alterations were quite heterogeneous in different hepatocytes and even within the same cell severely altered mitochondria were observed adjacent to normal ones (Figure 1; B, C, and G to J ▶ ). The mitochondria in sinusoidal cells (endothelial cells and Kupffer cells), in contrast to the ones in hepatocytes, appeared normal as did the mitochondria in fibroblasts of the sparse connective tissue of the fetal or newborn liver.

Mitochondrial Alterations Are also Present in Other Tissues and in Blood Cells

In addition to changes of the mitochondrial compartment in hepatocytes, structural alterations of the mitochondrial population of several lipid-metabolizing organs and tissues, which are also pathologically altered in Zellweger syndrome, were observed. In the epithelial cells of the adrenal cortex of PEX5^−/− mice, especially in the inner zona fasciculata, severely altered mitochondria were found (Figure 2, A and B) ▶ , showing either enlarged lamellated mitochondrial granules (Figure 2A) ▶ or accumulation of electron-dense material in the intermembrane space of tubular cristae (Figure 2B) ▶ . Focal proliferation of pleomorphic mitochondria with different types of lamellated granules were also seen in epithelial cells of proximal tubules of the kidney, known to be rich in peroxisomes (Figure 2, C and D) ▶ . In contrast, the mitochondrial compartment in other parts of the nephron was not altered. In cardiomyocytes of PEX5^−/− mice, routine electron microscopy revealed an increase in size and number of mitochondrial granules and the presence of megamitochondria (Figure 2F) ▶ . Pathological alterations in skeletal muscle were dependent on the region of the body and the specific muscles investigated. Most severe alterations were observed in the diaphragm, consisting of blebs on the surface of mitochondria (not shown) as well as rarefication and curvilinear alterations of mitochondrial cristae. In some myocytes of leg muscles subsarcolemmal aggregates of mitochondria and megamitochondria with bizarre forms were present (not shown). On the other hand, mitochondria in smooth muscle cells of the intestine displayed only slight alterations, which consisted either of the enlargement of the intermembrane space of the cristae (Figure 2G) ▶ and blebs on the surface, or invaginations into their matrix (Figure 2H) ▶ . In light of the hematopoietic capacity of the liver in newborn mice, mitochondrial alterations were also evaluated in blood cells present in this organ. Whereas mitochondria were completely normal in precursors of red blood cells, such as normoblasts (not shown), they were abnormal in a small portion of developing neutrophils and lymphocytes (Figure 2I) ▶ . The specific alterations of blood cells were independent of the severity of mitochondrial changes in neighboring hepatocytes and were rather cell type-specific. Whereas in neutrophilic granulocytes, mitochondria with circular cristae were frequently observed (Figure 2I) ▶ , the alterations in lymphocytes were more discrete and resembled those of smooth muscle cells showing enlargement of the intercristae space with formation of tubular cristae (not shown).

Finally, electron microscopic analysis of the neocortex of PEX5 knockout mice revealed no major ultrastructural changes in the mitochondria of neurons (not shown), despite the accumulation of different classes of lipids in those cells, as shown previously. 1

Damaged Mitochondria Are Removed from the Cytoplasm by Increased Autophagocytosis

In addition to ultrastructural changes of the mitochondria, we observed other signs suggestive of increased cellular toxicity and damage to mitochondria. Thus, many mitochondria were found in autophagic vacuoles of adrenal cortical cells, cardiomyocytes, and hepatocytes of PEX5^−/− mice (Figure 2, J to L) ▶ . Indeed, with cytochemical staining for acid phosphatase activity, large autophagic vacuoles were seen containing many mitochondria (Figure 2M) ▶ .

Reduction of Complex I Activity Is the Most Consistent Biochemical Finding in Mitochondria of Liver and Heart

Because the five multimeric complexes (complexes I to V) of the electron transport chain reside in the mitochondrial inner membrane and severe ultrastructural changes of this compartment were found in PEX5^−/− mice, we investigated the activities and the protein contents of the respiratory chain enzymes. Liver, displaying the strongest morphological alterations of mitochondria, and heart, with more subtle changes, were analyzed. In Table 1 ▶ the activities of mitochondrial respiratory chain enzymes in liver and heart of newborn mice, expressed relative to the activity of citrate synthase are summarized. In comparison to control levels, the activity in liver of newborn PEX5^−/− mice was reduced to 45% for complex I and to 65% for complex V, whereas complex IV activity was increased up to 180%. In contrast, no change was observed in the activities of complexes II and III. These differences in respiratory chain complex activities were already present at day 18.5 of embryonic development, except for complex V, which was only reduced by 25%, a statistically insignificant change (not shown).

In heart tissue (Table 1) ▶ , which showed only slight ultrastructural alterations, the differences in activities of the mitochondrial respiratory chain enzymes between control and PEX5^−/− mice were also less severe than in liver. Complex I activity of newborn PEX5^−/− mice was reduced to 65% of the control value, but there was no significant difference in E18.5 animals (data not shown). On the other hand, complex IV activity was 20% higher in heart of newborn PEX5^−/− mice, but this difference was not statistically significant.

Corresponding to the normal appearance of mitochondria in fibroblasts in primary cultures of PEX5 knockout mice (not shown) or in situ in various organs, complex I activity was not altered in fibroblast cultures of these mice (either immortalized fibroblasts or primary cultures of passage 3 or 4) (data not shown).

Western blot analysis of different subunits of respiratory chain complexes from liver mitochondria (Figure 3) ▶ revealed an estimated 50% reduction of the 39-kd subunit of complex I, a less-pronounced reduction of the core I subunit of complex III and subunit I of complex IV. In contrast the analyzed subunits of complexes II and V were not significantly altered (Figure 3) ▶ .

Proliferated Mitochondria Form Large Aggregates in Hepatocytes of PEX5^−/− Mice

In addition to electron microscopy, we used an extravidin/peroxidase kit to detect mitochondria by light microscopy, based on their high content of biotinylated proteins (Figure 4; A to D ▶ ). 46 The staining pattern corresponded to the intracellular distribution of mitochondria in electron microscopical preparations. In contrast to the granular particulate appearance of mitochondria, some of which were localized around glycogen deposits, in hepatocytes of control mice (Figure 4, A and C) ▶ , aggregates of proliferated mitochondria in subplasmalemmal regions were observed in E18.5 PEX5 knockout mice (Figure 4B) ▶ . The abundance and proliferation of the mitochondrial aggregates that filled up some hepatocytes was even more pronounced in newborn PEX5^−/− mice (Figure 4D) ▶ , which in addition exhibited reduced glycogen deposits.

Proteins of Respiratory Chain Complexes Are Heterogeneously Distributed and Focally Reduced in Hepatocyte Mitochondria of PEX5^−/− Mice

To provide further insight into mitochondrial alterations, the localization of different mitochondrial respiratory chain complexes was investigated by immunohistochemistry on paraffin sections (Figure 4; E to L ▶ ). Optimal light microscopical localization of proteins of respiratory chain enzymes in hepatocytes was observed only after antigen retrieval by 3 × 5-minute microwave irradiation of paraffin sections. The localization of complexes III to V was possible thereafter using our standard procedure. 27 However, localization of complexes I and II was only achieved by means of additional catalyzed reporter deposition with biotinylated tyramine (TSA, tyramide signal amplification). 28 By applying these methods, a marked intercellular heterogeneity in the distribution of the mitochondrial respiratory chain enzymes was observed in hepatocytes of E18.5 and P0.5 PEX5 knockout mice (shown only for E18.5 fetuses in Figure 4; F, and H to L ▶ ). The strongest reduction of overall staining intensity was seen in preparations for complex I (Figure 4, E ▶ versus F), but a reduction, albeit to a lesser extent, was also present in preparations for other respiratory chain complexes. Despite aggregation of proliferated mitochondria in most hepatocytes, the stain intensity was patchy showing heterogeneity in neighboring cells of PEX5^−/− mice (Figure 4; H to L ▶ ).

The intracellular heterogeneity of mitochondrial respiratory chain enzymes was further substantiated by electron microscopical localization of cytochrome c oxidase enzyme activity in the liver of PEX5^−/− mice (both in E18.5 and P0.5; Figure 5, B and C ▶ ). In contrast, mitochondria in hepatocytes of control animals exhibited a uniform distribution of the cytochrome c oxidase activity (Figure 5A) ▶ . Interestingly, the majority of ghost-like or severely altered mitochondria were either negative or exhibited low-cytochrome c oxidase activity (Figure 5B) ▶ . On the other hand, the mitochondria with parallel-stacked cristae in hepatocytes of PEX5^−/− mice showed very strong staining of their longitudinal cristae, indicating increased cytochrome c oxidase activity (Figure 5C) ▶ . Interestingly, despite the relatively subtle changes of mitochondrial morphology in cardiomyocytes of PEX5 knockout mice, strong heterogeneity of cytochrome c oxidase staining with negative organelles next to heavily stained ones was also found in the heart (Figure 5F) ▶ . On the other hand, a uniform cytochrome c oxidase-staining pattern was present in the diaphragm, despite the more severe alterations of the mitochondrial ultrastructure (Figure 5, G and H) ▶ .

The ultrastructural localization of the respiratory chain enzyme proteins in hepatocytes was only possible with the antibody against complex III, which also gave the best results in light microscopical preparations. The labeling of complex III (not shown) revealed a strong reduction (more than five times) in the mitochondria of PEX5^−/− mice, when the ratio of immunogold labeling was determined by morphometry in 500 mitochondria. Thus, in contrast to 20.8% of mitochondria in controls only 4.2% of them from PEX5^−/− mice were labeled with gold particles representing antigenic sites for complex III. In addition, a strong heterogeneity of labeling of mitochondria in neighboring hepatocytes of PEX5 knockout mice was observed. Similar to the cytochemical results for cytochrome oxidase, a clearly stronger labeling for complex III was observed in normal-appearing mitochondria or in mitochondria with stacks of parallel cristae whereas mitochondria with abnormal cristae or mitochondrial ghosts contained less gold particles.

ATP Levels and Redox State in Hepatocytes of PEX5 Knockout Mice

The question whether changes in the overall activities of complex I and complex V would lead to reduced ATP levels in hepatocytes of PEX5 knockout mice, was investigated by measuring the ATP content in the liver (Table 2) ▶ . The mean value obtained for PEX5^−/− mice (2.69 ± 0.02 μmol/g liver) was slightly higher than the control value (2.14 ± 0.18 μmol/g liver), indicating that there is no deficiency in the steady-state level of hepatic ATP in peroxisome-deficient mice.

Because lactate/pyruvate and β-hydroxybutyrate/acetoacetate ratios reflect the redox state in the cytoplasm and in the mitochondria of cells, respectively, the four compounds were measured in livers of newborn mice (see Table 2 ▶ ). They were all reduced in PEX5 knockout livers in comparison to controls (percentage of control levels: lactate, 25%; pyruvate, 71%; β-hydroxybutyrate, 8%; and acetoacetate, 29%). These low values are probably because of starvation of the peroxisome-deficient mice as a consequence of the severe hypotonia and may therefore invalidate the ratios of lactate/pyruvate and β-hydroxybutyrate/acetoacetate as indicators for respiratory chain function. Contrary to what one would expect in case of a deficiently functioning respiratory chain, both ratios were strongly reduced in PEX5^−/− mice (34 and 27% of control values).

Induction of MnSOD in Different Organs of PEX5 Knockout Mice Suggests an Increase of ROS

Because some of the features of mitochondria in PEX5^−/− hepatocytes were reminiscent of conditions associated with oxidative stress and because complex I deficiency is known to be accompanied by the production of ROS, 47 we looked for further indications of oxidative stress in the liver of peroxisome-deficient mice. Because in several experimental conditions associated with increased oxidative stress mitochondria express increased amounts of MnSOD protein, 47 in situ hybridization and immunocytochemical procedures were optimized to localize the mRNA and protein of this enzyme. A strong and reproducible immunohistochemical signal for MnSOD was obtained with a polyclonal anti-MnSOD antibody received through the courtesy of Professor Kato. 26 When compared to wild-type littermates, PEX5^−/− mice at E18.5 and older, showed strong increases of MnSOD mRNA and protein expression in their livers (Figure 6; A to F ▶ ). In addition to the alterations in liver, heterogeneous induction of the MnSOD mRNA and protein was also found in cardiomyocytes (Figure 6; I to L ▶ ).

Electron microscopical localization of MnSOD in liver sections embedded in Unicryl, revealed intra- and intercellular heterogeneous expression of this protein in mitochondria of hepatocytes (Figure 6, G and H) ▶ . Analysis of 200 mitochondria by morphometry revealed that the immunolabeling for MnSOD was increased in PEX5^−/− mice. Thus, in contrast to 45% labeling in control mice, 59% of mitochondria were labeled in PEX5 knockouts. Interestingly, in hepatocytes of PEX5^−/− mice, mostly the normal-appearing, or ring-shaped mitochondria were labeled with the MnSOD antibody, whereas severely altered mitochondria were unlabeled.

Despite clear evidence by in situ hybridization and immunocytochemistry (Figure 6) ▶ the analysis of MnSOD mRNA and protein by Northern and Western blotting revealed no significant alterations in liver tissue of newborn peroxisome-deficient mice as compared to wild-type controls (data not shown). However, a twofold increase in MnSOD transcripts was found in two PEX5-deficient mice that exceptionally survived for 2 days (not shown).

Severe Mitochondrial Alterations Are Found in PEX5^−/− Mice, Corroborating the Fine Structural Changes Reported in Patients with Zellweger Syndrome

In PEX5^−/− mice the most prominent mitochondrial alterations were found in hepatocytes, exhibiting heterogeneous, focal proliferation of pleomorphic mitochondria in some cells and involving all mitochondrial subcompartments (outer membrane, mitochondrial cristae, and matrix space). These alterations are already present in E18.5 PEX5^−/− fetuses but become more pronounced after birth (P0.5), occasionally leading to severe and degenerative mitochondrial ghost forms (Figure 1; H to J ▶ ). Alterations of the mitochondrial fine structure in Zellweger patients were already described in the original report on the absence of peroxisomes by Goldfischer and colleagues. 12 Those structural changes were, however, less severe in nature and consisted of irregular and twisted cristae, dilatation of the intracristae space, as well as occurrence of attenuated organelles, corresponding to the subtle changes in the livers of our E18.5 PEX5^−/− mice. In several subsequent ultrastructural studies, 20-22,24,48 mitochondrial alterations of varying types and severity were reported, although in a few cases there also were no changes noted. 49,50

In addition to the liver, changes of mitochondria in Zellweger patients have been reported in skeletal muscle. 51,52 Consistent with the finding of Sarnat and colleagues, 51 the diaphragm of the PEX5^−/− mice was more affected than muscles of the limbs, which is interesting, because respiratory failure could be partly responsible for the death of some patients and possibly also of our mice. Furthermore, PEX5^−/− mice showed abnormalities of mitochondria with different severity in other tissues known to be altered in Zellweger syndrome, such as epithelial cells of the inner zona fasciculata of the adrenal cortex and the renal proximal tubules, smooth muscle cells of the duodenum, cardiomyocytes, and some blood cells (developing lymphocytes and neutrophils). Although Zellweger patient fibroblasts have been extensively studied, mitochondrial alterations in those cells have not been observed, which is consistent with our observations in PEX5^−/− fibroblasts. Finally, despite the depositions of different lipid derivatives in brain, 1 no obviously altered mitochondria were found in neurons of the cortical plate of PEX5 knockout mice. Mitochondrial changes were also not observed in neurons of Zellweger patients 53-55 but were seen in astrocytes, ranging from severely altered, degenerative forms 12 to mitochondrial proliferation. 50 Because pathological malformations of the brain occur focally, additional studies of the brain in PEX5 knockout mice are necessary before definitive conclusions on mitochondrial alterations in this organ can be drawn.

Ultrastructural Alterations of Mitochondria Are Associated with Changes in Respiratory Chain Complexes

As shown by a combination of activity measurements and Western blot analysis of liver homogenates, immunocytochemical localization of the enzyme proteins, as well as enzyme cytochemical staining for cytochrome c oxidase, there are profound alterations in the activity and content of different respiratory chain enzymes in mitochondria of PEX5^−/− mice. The most consistent finding was a twofold reduction of NADH-ubiquinone oxidoreductase (complex I) activity that correlated with the reduced levels of a complex I subunit in liver samples of newborn PEX5^−/− mice (Table 1) ▶ . These results are in line with the studies of Goldfischer and colleagues, 12 who noted a 70% decrease in O₂ consumption in mitochondrial fractions of liver of Zellweger patients when the fractions were incubated with complex I-linked substrates. In contrast to Zellweger patients who also displayed a reduced activity of complex II, this activity was in the normal range in liver or heart homogenates of PEX5^−/− mice. For complex III, there was a marked heterogeneity in immunocytochemical staining despite unchanged levels of total enzyme activity in homogenates. Importantly, severely altered mitochondria seemed to contain much less complex III than less affected mitochondria. Enzyme histochemical staining for cytochrome c oxidase (complex IV) also revealed a heterogeneous staining of the mitochondrial cristae. In individual hepatocytes, ultrastructurally normal mitochondria and mitochondria with parallel stacks of cristae exhibited a strong cytochrome c oxidase activity, whereas mitochondria with altered cristae showed a significantly weaker staining with mitochondrial ghosts exhibiting only minimal staining. The elevation of complex IV activity observed in liver homogenates is not in contradiction with the reduction observed for other respiratory chain enzymes, because this protein is differently regulated. 56 Indeed, in some patients with a reduced activity of complex I, an elevation of the activity of complex IV has been reported. 57

On the other hand, a significant 35% reduction of complex V was found in liver homogenates of newborn PEX5 knockout mice, and this was also reflected in reduced and heterogeneous staining of complex V in light microscopic preparations. No evidence of uncoupling of the mitochondria in liver and heart of PEX5^−/− mice was obtained by enzyme histochemical staining for ATPase activity (M. Borgers, data not shown). This is in agreement with an earlier report on liver mitochondria of Zellweger patients, 48 although in skeletal muscle a slight uncoupling of mitochondria has been described. 52

Taken together, the ultrastructural alterations of liver mitochondria are accompanied by an altered activity and distribution of the mitochondrial respiratory chain complexes leading to a heterogeneous mitochondrial population with an overall decrease of complex I and complex V activities in liver of newborn PEX5-deficient mice.

Deficiency of the Respiratory Chain Enzymes Does Not Affect ATP Levels

Because the respiratory chain is responsible for the production of ATP by oxidative phosphorylation and the regeneration of NAD⁺, functional defects of respiratory chain enzymes (mostly complexes I and IV) are often accompanied by low ATP levels and increased ratios of lactate/pyruvate and β-hydroxybutyrate/acetoacetate. 58 Unfortunately, because of starvation of the newborn PEX5^−/− mice, all redox markers were strongly depleted, interfering with the proper assessment of the redox status. Nevertheless, steady-state ATP levels were not altered in PEX5^−/− mice as compared to control littermates (Table 2) ▶ , suggesting that despite the reduction of the activities of complexes I and V, the overall mitochondrial respiratory function of the whole hepatocyte population is still sufficient. These results are consistent with the so-called “threshold effect” 59 in patients with complex I deficiency, who may have normal ATP production despite severe reduction of complex I activity. 47,57

Indications Suggestive of Increased Oxidative Stress in PEX5^−/− Knockout Mice

Mitochondrial electron transport has long been recognized as a major source of superoxide (.O₂⁻) and H₂O₂. 60 The superoxide anion radical is generated by direct transfer of electrons from complex I or complex III to oxygen. Under physiological conditions, 2 to 3% of the consumed oxygen is converted to superoxide. To protect the cell from toxic injury, .O₂⁻ is converted to the less toxic H₂O₂ by MnSOD present in the mitochondrial matrix. This H₂O₂ may either diffuse out of mitochondria into the cytoplasm or may remain in the mitochondrial matrix and is reduced to H₂O by glutathione peroxidases of the appropriate cellular compartment. If the balance between the generation and scavenging of these ROS is disturbed, increased oxidative stress may develop in mitochondria, 58,61 leading to oxidation of lipids in membranes, of thiol-containing proteins, and of mitochondrial DNA.

The ultrastructural alterations in the mitochondrial populations of different organs of PEX5^−/− mice are suggestive of oxidative stress. These alterations, which have also been reported in mitochondrial respiratory chain disorders, include: 1) proliferation of pleomorphic mitochondria, 2) abnormal cristae (eg, parallel stacks, curvilinear forms), 3) fluffy matrix, and 4) swollen mitochondria. 62 Similar ultrastructural alterations have also been described in the liver in many other disease states associated with oxidative stress (eg, Reye’s syndrome, acute fatty liver of pregnancy) and intoxication with drugs (eg, peroxisome proliferators, chloramphenicol, nonsteroidal anti-inflammatory drugs) or toxins (ethanol, CCl₄, antimycin A, and rotenone). 58,63,64 In Wilson’s (copper intoxication), Parkinson’s, or Huntington’s diseases and Friedreich’s ataxia similar mitochondrial alterations have also been described. 58,65 The early stage alterations observed in Wilson’s disease are similar to the mitochondrial changes in E18.5 PEX5^−/− mice (except for crystalline inclusions). Interestingly, analogous mitochondrial alterations have been noted in iron overload, which is associated with substantial depletion of endogenous antioxidants and moderate lipid peroxidative damage. 66 Similar pathological alterations could play a role in the pathogenesis of the secondary mitochondrial changes in Zellweger syndrome, because iron overload and deposition have also been reported in several organs of Zellweger patients. 48,52,67

Ultrastructural alterations of mitochondria with circular cristae and dilation of intracristal spaces similar to those reported here were recently described in syncytiotrophoblastic cells of human placenta and it was suggested that they represented an adaptive response to oxidative stress because of elevation of oxygen tension at the end of the first trimester. 68 The further intensification of degenerative mitochondrial alterations in newborn (P0.5) PEX5^−/− mice in comparison to 18.5-day embryos would also support the notion that oxidative stress, because of elevation of oxygen tension after the birth, is responsible for the worsening of the mitochondrial changes in PEX5 knockouts.

Another sign of oxidative stress in liver mitochondria of PEX5^−/− mice was the significant up-regulation of MnSOD (Figure 6) ▶ , which is an important enzyme for the protection against the development of oxidative stress. 69,70 An increase in MnSOD was also reported in some patients with complex I deficiency. 47

As mentioned above, the sites of radical production in the respiratory chain are complexes I and III. A clear correlation between the degree of complex I inhibition, radical production, and cell injury by lipid peroxidation was shown in different experimental models. 71 Because in PEX5^−/− mice, complex I activity was twofold reduced in liver homogenates and complexes I and III showed a lower immunostaining intensity in liver sections, it is plausible that increased mitochondrial radical production occurs in the peroxisome-deficient mouse livers. Interestingly, it has been demonstrated that radicals produced in mitochondria are themselves effective inhibitors of different respiratory chain enzymes and that the pattern of inactivation is different for distinct oxygen radicals. 72 Thus, a vicious cycle could be initiated whereby the reduced activities of complex I and possibly complex III would release ROS that in turn could further inhibit the activities of the respiratory chain enzymes.

Origin and Consequences of Mitochondrial Changes

The fact that the absence of functioning peroxisomes is associated with an altered mitochondrial structure and function in man as well as in mice indicates that a conserved mechanism underlies these subcellular changes. In some Zellweger patients no mitochondrial alterations were observed, but this might be related to the genetic heterogeneity of the syndrome that can indeed be caused by defects in at least 10 different genes. 17 In PEX2-deficient mice, another model of Zellweger syndrome, 73 even though no clear-cut mitochondrial alterations in hepatocytes were reported, a few morphologically abnormal mitochondria can be identified in the published electron micrographs (eg, Figure 6B ▶ in Faust and Hatten 73 ).

In speculating on a link between the absence of peroxisomes and the mitochondrial alterations two possibilities must be taken into consideration which are not necessarily mutually exclusive: 1) defective rescue function of peroxisomes against ROS and 2) defective detoxification function of peroxisomes.

A physiological function of peroxisomes in defense against oxidative stress has been well established in plant cells. 74 Moreover, the localization in mammalian liver peroxisomes of various enzymes involved in generation and detoxification of ROS such as xanthine oxidase, 75 superoxide dismutases, 76, 77 and glutathione peroxidase 78 suggest strongly that they must be involved in regulation of cellular redox processes. 3 Indeed, Reddy and Lalwani 79 were the first to draw attention to the generation of ROS in hepatic peroxisomes of rodents treated with peroxisome proliferators and its possible implications for hepatocarcinogenesis. The same group described recently the importance of peroxisome proliferation in regulation of H₂O₂ levels in the liver of acyl-CoA oxidase I knockout mice. 80 Moreover, the complex interaction between the peroxisomal and mitochondrial lipid metabolism in the pathogenesis of fatty liver was shown in mice nullizygous for both peroxisomal acyl-CoA oxidase and PPAR-α. 81 In oxidative stress induced by ischemia and reperfusion of kidney, degenerative changes of regular peroxisomes in proximal tubules was noted by Singh 3 and Gulati and colleagues 82 that is followed by a compensatory regeneration of smaller particles. Interestingly, in the same experimental model there is evidence of severe reduction of complex I activity in renal mitochondria, 83 suggesting an association between peroxisome degeneration and mitochondrial complex I inactivation, similar to PEX5^−/− mice. Furthermore, fibroblasts from patients with peroxisome biogenesis disorders have been shown to be much more sensitive than controls to ROS generated by UV irradiation, 84,85 which has been suggested to be because of reduced plasmalogen levels, which might have an important anti-oxidative function in biomembranes. 86 Interestingly, HepG2 cells with a prominent peroxisome compartment 87 respond to UV irradiation by tubulation and proliferation of peroxisomes. 5 Finally, H₂O₂ was recently shown to induce several PEX genes that are involved in biogenesis of peroxisomes. 4 Taken together there is substantial evidence that peroxisomes have an important function in cellular rescue against ROS. How the cellular redox state is affected when both ROS-generating and -detoxifying enzymes are mislocalized to the cytoplasm such as in our PEX5 knockout mice, however requires further study. Nevertheless, it seems highly likely that the absence of peroxisomes leads to increased ROS levels in cells with prominent peroxisomal metabolism that in turn could impair the mitochondrial respiratory chain enzymes triggering a vicious cycle of mitochondrial degeneration as mentioned above.

An alternative explanation for the damage to the mitochondrial compartment might be the toxic effects of accumulation of compounds that are normally degraded by peroxisomal β-oxidation eg, very long-chain fatty acids, dicarboxylic acids, bile acid intermediates, and branched chain fatty acids. Notably, it was recently found that bile duct ligation in rats causes impairment of the mitochondrial respiratory chain, possibly by altering the lipid composition of the inner mitochondrial membrane. 88 Furthermore, it is well known that free fatty acids act as potent detergents that can damage cellular membranes. 89 In this respect it is important to mention that the mitochondrial changes in the PEX5 knockout mice are most prominent in tissues with an active lipid metabolism and a high content of peroxisomes (eg, liver, proximal renal tubules, and adrenals) and that the mitochondrial damage described in this report seems to be a cell autonomous phenomenon. The latter was exemplified in liver where liver parenchymal cells displayed severe mitochondrial changes whereas neighboring endothelial cells, Kupffer cells, and connective tissue cells did not show any abnormalities.

Interestingly, Mathis and colleagues 90 have noted that the mitochondrial changes in the liver of Zellweger patients were already present before the development of major hepatic disease. Indeed, in our newborn peroxisome-deficient mice, also no severe liver pathology was observed, suggesting that the mitochondrial alterations could play a role in the pathogenesis of the liver dysfunctions at later stages of the disease. In conclusion, the severely altered mitochondria in various organs of peroxisome-deficient mice that may be induced by oxidative stress could play a major role in the pathogenesis of the multiple organ dysfunctions observed in this devastating disorder.