Hypothalamic mitochondrial dysfunction associated with anorexia in the anx/anx mouse

Abstract

The anorectic anx/anx mouse exhibits disturbed feeding behavior and aberrances, including neurodegeneration, in peptidergic neurons in the appetite regulating hypothalamic arcuate nucleus. Poor feeding in infants, as well as neurodegeneration, are common phenotypes in human disorders caused by dysfunction of the mitochondrial oxidative phosphorylation system (OXPHOS). We therefore hypothesized that the anorexia and degenerative phenotypes in the anx/anx mouse could be related to defects in the OXPHOS. In this study, we found reduced efficiency of hypothalamic OXPHOS complex I assembly and activity in the anx/anx mouse. We also recorded signs of increased oxidative stress in anx/anx hypothalamus, possibly as an effect of the decreased hypothalamic levels of fully assembled complex I, that were demonstrated by native Western blots. Furthermore, the Ndufaf1 gene, encoding a complex I assembly factor, was genetically mapped to the anx interval and found to be down-regulated in anx/anx mice. These results suggest that the anorexia and hypothalamic neurodegeneration of the anx/anx mouse are associated with dysfunction of mitochondrial complex I.

The anorectic anx/anx mouse (1) is an attractive model for disturbed feeding behavior, as it exhibits phenotypes associated with failure to thrive in infants and young children (2), anorexia nervosa (3), and cachexia (4). This mouse arose by a spontaneous mutation (anorexia, allele symbol anx) and is characterized by poor appetite and reduced stomach content, and dies (likely because of the severe starvation) around 3 wk after birth (1). The anx/anx mice eat significantly less than their wild-type littermates, and by postnatal day (P) 21 they weigh half as much, rendering them an emaciated appearance (1). These mice also exhibit a number of neurological abnormalities, such as body tremors, head weaving, hyperactivity, and uncoordinated gait (1).

Several neurotransmitter (5, 6) and neuropeptidergic (7–11) systems involved in the regulation of food intake and energy metabolism are disturbed in the anx/anx mouse. The majority of these findings are centered around the hypothalamus, the origin of a neuronal network important for the control of initiation and termination of food intake, as well as diet-induced thermogenesis and energy expenditure. The arcuate nucleus of hypothalamus (Arc), situated at the interface between the periphery and brain, receives signals about energy status from the periphery, such as circulating leptin and insulin levels, resulting in anorexigenic or orexigenic behavior. Two main populations of food intake-regulating neurons reside in the Arc, both expressing leptin and insulin receptors (12). One group coexpresses the two orexigenic neuropeptides neuropeptide Y (NPY) and agouti-gene related protein (AGRP) (11, 13), whereas the other population expresses anorexigenic proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) (14). Both these groups undergo degeneration in the anorexic anx/anx mouse (15). By P21, anx/anx mice exhibit fewer hypothalamic fibers immunoreactive (ir) for AGRP and NPY (7, 8, 10, 11, 16), as well as α-melanocyte–stimulating hormone (αMSH) and CART (7, 9). In the anx/anx mouse, the orexigenic AGRP system develops normally until P12, after which the normal, continuous increase in AGRP-ir fiber density in the main projection areas ceases, and in some areas even decreases. These changes overlap both temporally and spatially with activation of microglia (10) and expression of MHC class I gene (H2-D_b) by both neurons and glia cells (15). In addition, we have shown increased amounts of apoptotic cells and presence of activated caspase 6 in NPY-ir fibers in the hypothalamus of these mice, indicating degenerative processes (15).

Interestingly, the anx/anx mouse shares many symptoms with mitochondrial oxidative phosphorylation system (OXPHOS) complex I (CI) deficiencies, including poor feeding, neurodegeneration, and muscle weakness (17), including the eyelid muscles (OMIM 252010). In the late stage of disease, the anx/anx mice keep their eyes partly shut, possibly because of weakening of the eyelid muscles. Taking these data together, we hypothesized that the starvation and neurodegeneration observed in the anx/anx mouse could be related to defects in the mitochondrial OXPHOS (17) and possibly CI deficiency.

In the present study, using Affymetrix microarray, we found that the most prominent cellular pathways likely to be affected in Arc of the anx/anx mouse include mitochondrial function and the OXPHOS. We therefore assessed the efficiency of OXPHOS and CI in anx/anx mice by high-resolution respirometry. The levels of fully and partly assembled CI were studied using native Western blots, and markers for reactive oxygen species (ROS) and oxidative stress were analyzed by histochemical methods. Mapping of the interval of the anx gene and mutation revealed that one of the CI assembly factors, the NADH dehydrogenase (ubiquinone) 1α-subcomplex, assembly factor 1 (Ndufaf1)-gene, is located in the interval. This gene was also found to be down-regulated in anx/anx mice, specific for the anx-allele.

Results

Mitochondrial CI Deficiency in the anx/anx Mouse Hypothalamus.

A microarray analysis of Arc from anx/anx (n = 3) and +/+ (wild-type) (n = 3) mice revealed 132 up-regulated genes and 73 down-regulated genes in anx/anx mice, with ≥ 1.4-fold average increase or decrease (Table S1). Through an Ingenuity Pathway Analysis (IPA) of these genes, we identified the top canonical pathways most likely to be involved in the phenotype of the anx/anx mice. The pathway analysis identified several genes related to OXPHOS pathways and mitochondrial functions (Fig. S1 and Table S2). Genes related to mitochondrial function and oxidative stress (e.g., Sod1, Prdx1, Bcl211, and Cox5B) are shown in Table 1. In addition, the top canonical pathways identified included functions, such as cell death and morphology, cellular growth, and proliferation and inflammatory responses (Table S2).

Table 1.

Selected mitochondria and oxidative stress-related genes showing altered expression when comparing anx/anx with +/+ mice

Symbol	Full gene name	Fold-change
Bcl2l11	BCL2-like 11 (apoptosis facilitator)	2.1
Atf4	Activating transcription factor 4 (tax-responsive enhancer element B67)	2.0
Gstm5	GST mu 5	1.8
Sod1	Superoxide dismutase 1, soluble	1.7
Ndufa5	NADH dehydrogenase (ubiquinone) 1 α subcomplex, 5, 13 kDa	1.7
Cox6a1	Cytochrome c oxidase subunit VIa polypeptide 1	1.6
Ubb	Ubiquitin B	1.6
Cox4i1	Cytochrome c oxidase subunit IV isoform 1	1.6
Cox5b	Cytochrome c oxidase subunit Vb	1.5
Prdx1	Peroxiredoxin 1	1.5
Cox6c	Cytochrome c oxidase subunit VIc	1.5
Ndufc2	NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 2, 14.5 kDa	1.5
Cox17	COX17 cytochrome c oxidase assembly homolog (Saccharomyces cerevisiae)	1.4
Gpd2	Glycerol-3-phosphate dehydrogenase 2 (mitochondrial)	−1.4
Map2k4	Mitogen-activated protein kinase kinase 4	−1.6
Atp5f1	ATP synthase, H+ transporting, mitochondrial F0 complex, subunit B1	−2.2
Ndufaf1	NADH dehydrogenase (ubiquinone) 1 α subcomplex, assembly factor 1	−2.2

Because both the phenotype and the microarray results suggest hypothalamic mitochondrial dysfunction in the anx/anx mouse, we assessed mitochondrial respiration in mechanically permeabilized hypothalami from anx/anx and +/+ mice (n = 6–9 per genotype) using high-resolution respirometry (Fig. 1A). Oxygen flux normalized to hypothalamus wet weight was significantly reduced in anx/anx mice compared with +/+ littermates. The anx/anx mice showed a 40% reduction in oxygen consumption, when mitochondrial respiration was assessed at the leak state by addition of CI substrates, malate and pyruvate (ADP not added). We could also detect a significant reduction (29%) in CI respiration in anx/anx mice, when the oxidative phosphorylation system was evaluated after the subsequent addition of ADP and glutamate (coupled respiration from CI), and after the addition of the exogenous uncoupler carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) to test the capacity of the electron transfer system I (ETSI). To discern whether the defects observed in the OXPHOS were specific for CI or a more general defect of the respiratory chain, we evaluated mitochondrial respiration through CII by addition of succinate. We did not find any significant differences between anx/anx and +/+ mice, when oxygen consumption was measured after this addition, neither in the coupled state (CIIa) nor after the addition of the exogenous uncoupler FCCP (ETSII), suggesting a specific CI defect. In other words, if the defect observed in mitochondrial respiration was because of a malfunction of complex II, III, IV, or V, we should have observed a decrease in oxygen consumption also after succinate addition. However, the increase in oxygen consumption generated by succinate addition was the same in anx/anx and +/+ mice, both in the coupled state (CIIa) and after addition of FCCP and inhibition of CI by rotenone (ETSII). To ascertain that the differences in the OXPHOS system in anx/anx mice were not related to an abnormal amount of mitochondria, we calculated the proportions of mitochondrial (cytochrome B and cox1) vs. nuclear (cyclophyllin) DNA with SYBR green real-time PCR. We found no difference between anx/anx (n = 4) and +/+ (n = 4) hypothalami (Fig. S2).

Fig. 1.

Hypothalamic complex I deficiency and mitochondrial dysfunction in the anx/anx mouse. Complex I respiratory capacity (A) is impaired in anx/anx mice. Mitochondrial respiration was analyzed in hypothalamus samples (n =6–9 per genotype). For basal O₂ flux from CI of the respiratory chain, malate and pyruvate were added to the Oxygraph-2k chamber (Leak). To maximize oxidative phosphorylation from CI, ADP and glutamate were added in sufficient amounts and in subsequent steps to couple electron transfer to ATP production (i.e., coupled respiration from CI). To ascertain O₂ flux from CI and CII, succinate was added (CI+II). We calculated additional O₂ flux through complex II, subtracting CI from CI+II (CIIa). To reach maximal capacity of the electron transfer system by CI and CII, the uncoupler FCCP was titrated to the chamber (ETSI+II). CI was inhibited by the addition of rotenone (ETSII). Electron transfer trough CI was calculated by subtracting ETSII from ETSI+II.The remaining O₂ flux after inhibition of CIII with antimycin A (O₂ flux independent of the electron transfer system) was subtracted from each of the previous steps. Error bars represent SE of mean. BN analysis (B) revealed lower levels of fully assembled CI in the hypothalamus of anx/anx mice (n = 3, exemplified by lanes 2 and 4) compared with +/+ mice (n = 4, exemplified by lanes 1 and 3) and CIII, but the levels of levels of CII and CIV were unchanged. In-gel activity assay (C) revealed lower activity of fully assembled CI in anx/anx mice (n = 4, exemplified by lane 2) compared with +/+ mice (n = 3, exemplified by lane 1). Abbreviations are listed in Table S4. *P < 0.05.

To evaluate if the decreased CI activity was caused by abnormal amounts or assembly of CI, we performed 1D blue native-polyacrylamide gel electrophoresis (BN-PAGE) to study the assembly pattern of the different mitochondrial complexes in anx/anx hypothalamus. This analysis showed lower levels of fully assembled CI in the anx/anx hypothalamus compared with +/+ mice, revealed by reduced amounts of protein complexes of ∼700 kDa, with CI-specific activity (0.66 vs. 1.64, P = 0.04; n = 10) (Fig. 1 B and C). The levels of CIII were also decreased in anx/anx compared with +/+ hypothalamus (0.56 vs. 1.7, P = 0.02; n = 10) (Fig. 1B). However, the amount of CII, CIV, and CV were unchanged (CII: 0.95 vs. 1.2, P = 0.4; n = 10; CIV: 1.0 vs. 1.2, P = 0.7; n = 6; CV: 0.85 vs. 1.25, P = 0.5; n = 4) (Fig. 1B). The in-gel staining confirmed CI activity of the 700-kDa band and revealed lower activity of CI in the hypothalamus of anx/anx (n = 4) compared with +/+ (n = 3) mice (9.7 vs. 14.4, P = 0.01) (Fig. 1C).

Increased Hypothalamic ROS Production and Signs of Oxidative Stress in the anx/anx Mouse.

CI deficiency is often accompanied by oxidative stress (18). We therefore measured hypothalamic ROS production in anx/anx and +/+ mice using intravenous dihydroethidium (DHE) injections. Microscopic analysis revealed small and dot-like red fluorescent staining, indicating ROS, to a higher number in anx/anx (n = 3) (Fig. 2 A, B, and D) than in +/+ (n = 3) Arc at P21 (Fig. 2C) (4.1 vs. 2.2, P < 0.05). The red fluorescent staining was localized to the area harboring the Arc AGRP/NPY and POMC/CART neurons and occasionally appeared to be localized within neurons, as seen in Y1- (Fig. 2B) and NeuN-positive (Fig. S3) cells, as well as in microglia (Fig. 2D).

Fig. 2.

ROS and SOD2 in anx/anx hypothalamus. Micrographs show ROS staining and immunofluorescent labeling for AGRP (A), Y1 (B and C), Iba1 (D) in Arc of anx/anx (n = 3, A, B, and D) and +/+ mice (n = 3, C) at P21. Immunofluorescence micrographs show SOD2 (E–G) and AGRP (E and F) in Arc of an anx/anx mouse (n = 4, E and G) and absence of staining in +/+ mouse (n = 4, F), both at P21. Arrows in A and B indicate ROS-staining; arrowhead in D indicates possible ROS staining within Iba1⁺ cell. Arrowhead in E indicates a SOD2⁺ neuron also expressing AGRP. Asterisk in G marks a small part of a section of an adjacent brain that was mounted and cut together with the anx/anx brain. [Scale bar: 50 μm in A (applies to A–C); 10 μm in D; 20 μm in F (applies to E and F); 200 μm in G.] Abbreviations are listed in Table S4.

Furthermore, immunohistochemistry (IHC) staining for superoxide dismutase 2 (SOD2), a major ROS-scavenging antioxidant in mtochondria (19), revealed increased immunoreactivity in Arc of anx/anx mice at P15 and P21 (n = 4 per genotype and age) (Fig. 2 E–G). In anx/anx, 12.5% of the Arc area was covered by SOD2-ir, versus 5.0% in +/+ (P = 0.05), indicating increased oxidative stress in the anx/anx Arc. On some occasions we found SOD2⁺ cells in Arc that also expressed AGRP (Fig. 2E), although this peptide could not be detected in the majority of the SOD2⁺ cells. We never found SOD2⁺ cells that convincingly expressed β-endorphin, a marker for Arc POMC/CART neurons. Occasionally, low-intensity SOD2 labeling was also seen in anx/anx mice at P10 and in +/+ mice of all ages. Preadsorption of SOD2 antiserum with the immunogenic SOD2 fragment resulted in a virtually complete disappearance of immunoreactivity.

IHC Analysis of Substantia Nigra in the anx/anx Mouse.

As the anx/anx mice display motor disturbances, and because CI inhibition—as well as oxidative stress—have been implicated in the pathogenesis and degeneration of nigral dopaminergic neurons in Parkinson disease (20), we investigated the prevalence of neurodegenerative and neuroinflammatory processes in the substantia nigra (SN) of anx/anx mice. Degenerative processes in the SN can be expected to cause an increased activation of microglia, revealed by increased ionized calcium-binding adapter 1 (Iba1)-labeling of bushy-shaped microglia, as seen in several hypothalamic areas of the anx/anx mice (10). However, no obvious increase was seen in anx/anx mice after Iba1 staining. Neither did IHC analysis of cholecystokinin (CCK) and tyrosine hydroxylase (TH), two markers expressed in mesencephalic dopamine neurons (21), reveal any apparent deviation in the staining pattern in anx/anx compared with +/+ mice (n = 2 per genotype).

Identification of Ndufaf1 as an anx Candidate Gene.

To determine the genetic cause behind the phenotype of the anx/anx mouse, we set up two intercrosses to genetically map the anx gene and mutation. The anx gene was previously mapped to ∼20 cM proximal of the nonagouti locus on mouse Chromosome (Chr) 2 (1). In the present study, a total of 4,844 meioses from the two different intercrosses identified a number of recombination events on both sides of the anx locus. The anx locus was thus mapped to a 0.2-cM interval, between markers D2Mit133 and Jojo5 (Chr 2: bp 118,889,896–120,175,108; www.ensembl.org) (Fig. S4). One recombination event in an F2 progeny from cross 2 (B6C3Fe-anx A/+ a × CAST/Ei) identified D2Mit133 as the proximal flanking marker. Three recombination events in three F2 offspring from cross 1 (B6C3Fe-anx A/+ a × B6C3H F1) identified Jojo5 as the distal flanking marker. Both flanking markers were supported by a number of recombination events on each side of the anx interval in both crosses. The anx mutation was found to cosegregate with markers D2Mit104, D2Mit395, and Jojo8 (Fig. S4).

The microarray analysis of Arc from anx/anx and +/+ mice identified one of the 73 down-regulated genes as the CI assembly factor Ndufaf1 (−2.220 fold-change). This gene was also mapped to the anx gene interval (Chr 2: bp 119,481,182–119,488,563 reverse strand; www.ensembl.org), and was thus considered a strong anx-gene candidate. However, sequencing of Ndufaf1, both genomic (exons) and cDNA, revealed no unique alterations in the anx/anx mice (n = 3); thus the mutation does not appear to be located in any of the coding regions of the Ndufaf1 gene.

The Ndufaf1 down-regulation was confirmed by Taqman real-time PCR. In the anx/anx brain, liver, and lung the Ndufaf1 mRNA levels were approximately half of +/+, whereas in pancreas the difference was even larger, down to 20% (n = 6 for each genotype and tissue) (Fig. 3A). No significant difference in Ndufaf1 expression was found in the heart or kidney.

Fig. 3.

Relative expression levels of Ndufaf1 in anx/anx and +/+ mice (A), normalized against two endogenous controls (n = 6 for each genotype and tissue). Because of the large intertissue variation, all values were also normalized against the +/+ mean. Exact P values (normalized against): brain (B) P = 0.07, (G) P = 0.003; pancreas (B) P = 0.03, (T) P = 0.00004; muscle (P) P = 0.003, (T) P = 0.06; lung (T) P = 0.003, (B) P = 0.008; liver (B) P = 0.1; (T) P = 0.002. Error bars represent ranges, adjusted for the normalization against the +/+ mean. *P ≤ 0.05; **P ≤ 0.01. Western blot (B) revealed down-regulation of Ndufaf1 in brain of anx/anx mice (n = 9, lane 1) compared with +/+ mice (n = 9, lane 2). (C) Quantification of Ndufaf1 Western blot. ***P ≤ 0.001. B, β-actin; G, GAPDH; P, PPIA; T, TATAbp. Abbreviations are listed in Table S4.

To determine if the reduced Ndufaf1 expression was linked to the anx allele, we analyzed the expression levels of wild type (WT) and anx allele expression of Ndufaf1 in four heterozygous mice, using a silent T/C SNP to separate the two alleles. The ratio between anx and WT alleles [(peak height_{anx allele} [cDNA]/ peak height_{wt allele} [cDNA])/(peak height_{anx allele} [gDNA]/ peak height_{wt allele} [gDNA])] were in all four instances lower than 1, indicating a lower expression of the anx allele (Table 2). On average, we detected an ∼22% reduction associated with the anx allele compared with WT in heterozygous mice (P = 0.015; n = 4), suggesting a 44% reduction of the Ndufaf1 transcript in homozygous anx/anx mice, in accordance with the twofold reduction detected by microarray and real-time PCR. This result thus indicates that the down-regulation of Ndufaf1 expression in anx/anx mice is indeed associated with the anx allele of Ndufaf1, rather than a secondary effect of the phenotype. In addition, Ndufaf1 and RNA polymerase II associated protein 1 (Rpap1) were the only genes in the anx interval that showed altered expression (both down-regulated) in the microarray analysis. Ndufaf1 and Rpap1 are separated by four genes with no observed alterations in expression and ∼10 Mb, indicating that it is not a region-specific down-regulation. Rpap1 was not included in the top canonical pathways, identified by IPA, and was thus regarded unlikely to be involved in the phenotype of the anx/anx mouse.

Table 2.

Allele-specific expression of anx and WT alleles in heterozygous mice analyzed by pyrosequencing

Mouse ID	anx allele/WT allele (cDNA)	anx allele/WT allele (gDNA)	anx allele/WT allele (cDNA/gDNA)*
2602	0.88	1.10	0.80
2618	0.88	1.04	0.85
2623	0.89	1.38	0.65
2626	0.87	1.08	0.81

*P = 0.0015, as calculated by one-sample t-test [mean value anx allele/WT allele (cDNA/gDNA) = 0.7775].

Western blot analysis showed that the down-regulation of the Ndufaf1 mRNA is also accompanied by an ∼50% decrease (0.8 vs. 1.3, P < 0.001) of the corresponding protein level in the anx/anx brain (Fig. 3 B and C). The analysis was repeated three times, including three mice for each genotype, and a similar decrease was always observed. Actin was used as a loading-control and revealed no difference between samples (Fig. 3 B and C).

Discussion

There are a number of possible explanations for the anorectic phenotype in the anx/anx mouse (1) as well as for the aberrant appearance and degeneration of the arcuate food intake-regulating circuitries in this mouse (5, 7–10, 16). Here we provide evidence for mitochondrial dysfunction in the hypothalamus of the anx/anx mouse. More specifically, we have detected a reduction in the CI assembly factor Ndufaf1 expression and lower levels of fully assembled CI, accompanied by a 29% reduction of CI-specific respiration efficiency in the anx/anx mouse. This finding is in agreement with previous findings, showing that reduction of Ndufaf1 mRNA and protein levels results in decreased CI assembly and activity (22). We also detected a decrease in CIII levels, but not in CII, CIV, and CV. The reason why CIII also show reduced levels in anx/anx hypothalamus is not understood. However, there are reports on mutations affecting CI that also significantly reduce the amounts of CIII and CIV (23–25), which is in accordance with the theory that CI, CIII, and CIV form supercomplexes to reduce the diffusion distance of the substrates, and thereby are able to affect the stability of each other (26, 27). Another possible explanation is that the increased ROS production observed in the anx/anx hypothalamus is influencing the CIII assembly.

The OXPHOS of the mitochondria is the major site for ATP production in cells, and CI is the largest of the five protein complexes (CI–CV) of this system. CI oxidizes NADH and thereby releases electrons that are transferred via the electron carriers ubiquinone and cytochrome c, as well as CIII and CIV, to the final acceptor, molecular oxygen. The energy generated by this transfer is used to build up the electrochemical gradient necessary for generation of ATP by CV (ATP synthase). In addition, CI is the main intracellular source of ROS in the mammalian cell during normal conditions, by “leaking” electrons instead of transferring them to the electron carriers. Even under normal conditions it is estimated that ∼2% to 5% of the oxygen consumed by mitochondria are converted to O₂^•− (28). ROS are therefore natural byproducts of the normal oxygen metabolism and have several roles in cell and metabolic signaling (24, 25). However, ROS production can increase dramatically during different conditions, such as CI dysfunction (23), resulting in oxidative stress and thereby significant damage of cell structures, eventually contributing to cell death (29). This finding is in agreement with our findings of increased levels of ROS in the hypothalamus of the anx/anx mouse. Furthermore, we show increased SOD2 immunoreactivity specifically in Arc of the anx/anx mouse, providing indications of oxidative stress, which is possibly caused by the increased levels of ROS, as SOD2 is a major ROS-scavenging antioxidant in mitochondria (19). The microarray performed in this study also shows that several other genes related to oxidative stress responses (e.g., the ROS scavengers Sod1, Prdx1, Bcl211, and Cox5B) are up-regulated in the anx/anx mouse Arc (Table 1).

Increased ROS levels have been implicated in the pathology of several diseases, such as cancer, atherosclerosis, inflammation, and neurodegenerative disorders, and have been shown to induce apoptosis or necrosis (18). However, ROS may also act as a metabolic signaling molecule, in addition to being an oxidative stressor (30). For example, ROS-signaling pathways play a significant role in hypothalamic metabolic sensing of both glucose and lipids (31, 32). Thus, high glucose concentrations increase hypothalamic ROS production, and ROS influence neuronal activation of Arc neurons. Hypothalamic ROS are also essential for the reduced food intake observed in intralipid-injected rats (31). In addition, uncontrolled generation of ROS in NPY/AGRP neurons causes impaired firing of these neurons (33, 34). Consequently, the increased hypothalamic ROS levels that we have observed in the anx/anx mouse could be causally involved in the initial starvation and anorectic phenotype of this model. In the longer perspective, a chronic overproduction of ROS could lead to the signs of oxidative stress and neuronal degeneration of both the orexigenic and anorexigenic arcuate neurons that we have previously observed in the anx/anx mouse (7, 15). In addition, the canonical pathways generated in our microarray analysis suggest that cell death and inflammatory processes occur in the hypothalamus of the anx/anx mouse. This finding is in agreement with previous studies suggesting inflammation and cell death in the anx/anx hypothalamus (10, 15, 35, 36).

The anx locus is still unknown. However, the present study suggests that the Ndufaf1 gene is equivalent to the anx gene. We have mapped the anx locus to a 0.2-cM interval on Chr. 2, containing more than 40 genes, including the CI assembly factor Ndufaf1. Thus, Ndufaf1 is approximately twofold down-regulated in anx/anx mice as shown by Affymetrix microarray analysis, real-time PCR, and Western blots. Furthermore, down-regulation of Ndufaf1 expression in anx/anx mice is associated with the anx allele of Ndufaf1, rather than being a secondary effect of the phenotype. Sequencing of the coding parts of the Ndufaf1 gene did not identify the anx mutation, suggesting that it is located in a regulatory element, such as the promoter-region or enhancers. Unfortunately, we have not been able to identify the promoter region of Ndufaf1, using standard in silico methods to reveal conserved regions among different species. In addition, this region on Chr. 2 is very gene-dense, and it is possible that Ndufaf1 enhancers are located within or in between other genes in the interval, thus making it difficult to identify them and evaluate the effect of possible sequence alterations. Further analysis of the 1.3-Mb anx-interval by resequencing is likely needed to identify the anx-mutation. In addition, further studies of, for example, Ndufaf1 knockout mice or transgenic rescue of anx/anx mice are needed to finally prove that Ndufaf1 is indeed the anx gene.

Why the phenotypic core features of the anx/anx mouse are related to brain functions remains to be answered, as well as why only certain neurons seem to be affected by the Ndufaf1 down-regulation in the anx/anx mouse even though Ndufaf1 is down-regulated in the whole brain as well as other tissues. One explanation could be related to a mutation located in a tissue/cell-specific promoter or other regulatory elements. Furthermore, various tissues—and possibly also cell-types—may have different demands on OXPHOS-derived energy, and the tissue/cell-specific need of CI activity may therefore vary (37, 38). In addition, the development of more subtle or slowly developing symptoms in less energy-requiring tissues/cells may be hidden behind the rapid development of more severe phenotypes in tissues/cells with high energy demand, such as brain and muscle. Interestingly, CI inhibition has long been implicated in the pathogenesis and degeneration of dopaminergic neurons of Parkinson disease (20). Thus, chronic infusion of low doses of the CI inhibitor rotenone induces selective dopaminergic neurodegeneration and Parkinson-like symptoms in rats, although the infusion causes a uniform CI inhibition throughout the brain (39). Moreover, selective neuronal damage and gliosis, as observed in the anx/anx mouse (10), seem to be common features in both human disorders with CI deficiencies (38) and animal models of CI-deficiency, such as the Ndufs4 knockout mouse (40). This finding suggests that different brain areas/neurons, with different energy demands, are more or less sensitive to CI deficiency and oxidative stress, possibly resulting in the selective neuronal damage seen in both animal models and human patients with CI deficiencies.

During the early postnatal period the Arc neurons differentiate into their adult state (41–43) and they are highly active and thus probably very energy demanding (44). Our hypothesis is that the Arc NPY/AGRP and POMC/CART neurons are more sensitive to even a moderate CI deficiency, and more prone to selective degeneration, during the first 3 postnatal weeks, when the anx/anx mice develop their symptoms. Because the dopaminergic subpopulation of neurons in SN projecting to striatum also have been shown to be sensitive to CI deficiency, and in view of the motor disturbances seen in the anx/anx mouse (1), we studied SN of anx/anx and +/+ mice by IHC. No apparent difference was detected with any of the markers used, Iba1, CCK, and TH, but it is possible that changes could be detected in SN with other markers and by using more sensitive approaches (e.g., stereology), or if the anx/anx mice would survive longer. It should also be mentioned that altered dopaminergic transmission has indeed been detected in the striatum of the anx/anx mice (6).

To what extent hypothalamic neurodegeneration occurs in rotenone-treated rodents remains to be explored. It is possible that the Arc neurons are less sensitive to CI-inhibition by, for example rotenone, after the first 3 postnatal weeks, when they have differentiated into adult state. Interestingly, selective atrophy of, among other parts, the hypothalamus and the striatum was reported in a patient with a null mutation in the NDUFA12L gene, encoding another assembly factor for CI (45), indicating that the hypothalamus could indeed be sensitive to CI deficiency in humans.

Like the anx/anx mouse, children with CI deficiencies are often born normal and develop disease during the first year of life (38), indicating that the organism is protected during the prenatal period. However, there is evidence for a markedly increased demand of OXPHOS-derived energy postnatally (45, 46). Interestingly, Dunning et al. described an 11-mo-old baby, who showed markedly reduced food intake, with two heterozygous mutations in the NDUFAF1 gene (47). These mutations resulted in reduced CI activity, similar to what we have observed in the anx/anx mouse.

The process of inactivated appetite-regulating neurons and subsequent degeneration, observed in the anx/anx mouse, may occur also in some human conditions. We hypothesize that a subclinical CI deficiency could become overt in the presence of cellular stress during, for example, severe starvation. This could lead to increased oxidative stress and possibly cause irreversible damage of the sensitive hypothalamic neurons, resulting in permanent damage of the appetite-regulating neuronal networks. Further studies on patients with such conditions are needed.

Materials and Methods

Animals.

Animal experiments were approved by the Stockholms Norra Djurförsöksetiska Nämnd ethical committee. Heterozygous anx breeding pairs (B6C3Fe–a/a–anx A/+ a) were originally obtained from The Jackson Laboratory. For more information, see SI Materials and Methods.

Microarray Analysis and IPA.

Total RNA was prepared, cRNA synthesized from anx/anx and +/+ basal hypothalamus, mainly Arc, and hybridized to an Affymetrix Mouse Genome 430 2.0 Array (Affymetrix Inc.). For information regarding data analysis and details, see SI Materials and Methods.

Oxygraph Measurements.

Mitochondrial oxygen consumption of anx/anx and +/+ hypothalami was assessed by high-resolution respirometry (Oxygraph-2K; Oroboros Instruments), in particular the consumption via CI and CII with slight modification of protocols previously described (48). For details, see SI Materials and Methods.

Mitochondria Count.

Real-time PCR with SYBR green was used to quantify the amount of mitochondrial DNA (cytochrome B and cox1) /genomic DNA (cyclophyllin) in hypothalami from anx/anx and +/+ mice using the Sequence detector system ABI-prism 7000 (Applied Biosystems Inc.). For details see SI Materials and Methods.

BN-PAGE and in-Gel Activity Assay.

Crude hypothalamic mitochondria were isolated from anx/anx and +/+ mice as previously described (49), followed by either Western blotting, to detect CI, CII, CIII, CIV, CV, and TOM40, or CI in-gel activity staining. For details, see SI Materials and Methods.

DHE Injections.

Intravenous injection of DHE (Invitrogen) was used to measure hypothalamic ROS production in anx/anx and +/+ mice. See SI Materials and Methods for details.

Immunohistochemistry.

IHC was performed as described previously (8). See SI Materials and Methods for detailed description and antibody information.

Mapping of the anx Gene.

Two intercrosses were set up to map the anx interval: cross 1 (B6C3Fe-anx A/+ a × B6C3H F1) and cross 2 (B6C3Fe-anx A/+ a and CAST/Ei). For information on genetic markers, PCR conditions, and calculation of recombination frequencies, see SI Materials and Methods.

Sequencing of Ndufaf1.

To identify Ndufaf1 sequence alterations between anx/anx and +/+ mice, genomic DNA and cDNA were prepared by standard procedures, followed by PCR amplification and sequencing using ABI 3730 DNA Analyzer and BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems). For more information on DNA/RNA preparation, PCR, sequencing conditions, and analysis see SI Materials and Methods.

Real-Time PCR.

Total RNA was prepared from seven different tissues, and cDNA was synthesized using standard methods. Samples were analyzed for expression of Ndufaf1 with Taqman real-time PCR (7900 HT Fast Real-time PCR system, Applied Biosystems Inc.) For details see SI Materials and Methods. Assays are listed in Table S3.

Allele Specificity.

A silent T/C SNP (Ensembl gene ID ENSMUSG00000027305 Ndufaf1 +87 T/C) was used to separate the expression level of the anx-allele from the WT-allele. DNA and cDNA of the two alleles from heterozygous anx/+ mice were PCR amplified over the T/C SNP and quantified using PyroSequencing (PSQ 96; Qiagen). Imbalanced expression of the two alleles was assessed by comparing the ratio of mean peak heights from the two alleles in cDNA to corresponding peak heights in genomic DNA, as described in SI Materials and Methods.

Western Blot Analysis.

Protein expression of Ndufaf1 was analyzed in total brain from anx/anx and +/+ mice according to standard procedures described in SI Materials and Methods.