Hypothalamic mitochondrial dysfunction associated with anorexia in the anx/anx mouse Charlotte Lindforsa,b,1, Ida A. K. Nilssona,b,c,1,2, Pablo M. Garcia-Rovesd,e, Aamir R. Zuberif, Mohsen Karimib,g, Leah Rae Donahuef, Derry C. Roopenianf, Jan Mulderh,i, Mathias Uhlénj, Tomas J. Ekströmb,g, Muriel T. Davissonf, Tomas G. M. Hökfeltc,i,2, Martin Schallinga,b, and Jeanette E. Johansena,b Departments of aMolecular Medicine and Surgery and gClinical Neuroscience, Karolinska Institutet, 171 76 Stockholm, Sweden; bCenter for Molecular Medicine, Karolinska University Hospital, 171 76 Stockholm, Sweden; Departments of cNeuroscience and dPhysiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden; eDiabetes and Obesity Laboratory, Institute for Biomedical Research August Pi i Sunyer and Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders, 08036 Barcelona, Spain; fThe Jackson Laboratory, Bar Harbor, ME 04609; hEuropean Neuroscience Institute at Aberdeen, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom; iScience for Life Laboratory, 171 21 Stockholm, Sweden; and jDepartment of Biotechnology, AlbaNova University Center, 106 91 Stockholm, Sweden Contributed by Tomas G. M. Hökfelt, September 16, 2011 (sent for review August 23, 2010) The anorectic anx/anx mouse exhibits disturbed feeding behavior fibers immunoreactive (ir) for AGRP and NPY (7, 8, 10, 11, 16), and aberrances, including neurodegeneration, in peptidergic neu- as well as α-melanocyte–stimulating hormone (αMSH) and anx/anx rons in the appetite regulating hypothalamic arcuate nucleus. Poor CART (7, 9). In the mouse, the orexigenic AGRP system feeding in infants, as well as neurodegeneration, are common develops normally until P12, after which the normal, continuous increase in AGRP-ir fiber density in the main projection areas phenotypes in human disorders caused by dysfunction of the ceases, and in some areas even decreases. These changes overlap mitochondrial oxidative phosphorylation system (OXPHOS). We both temporally and spatially with activation of microglia (10) therefore hypothesized that the anorexia and degenerative phe- and expression of MHC class I gene (H2-Db) by both neurons notypes in the anx/anx mouse could be related to defects in the and glia cells (15). In addition, we have shown increased OXPHOS. In this study, we found reduced efficiency of hypotha- amounts of apoptotic cells and presence of activated caspase 6 in lamic OXPHOS complex I assembly and activity in the anx/anx NPY-ir fibers in the hypothalamus of these mice, indicating de- mouse. We also recorded signs of increased oxidative stress in generative processes (15). anx/anx NEUROSCIENCE anx/anx hypothalamus, possibly as an effect of the decreased Interestingly, the mouse shares many symptoms with mitochondrial oxidative phosphorylation system (OXPHOS) hypothalamic levels of fully assembled complex I, that were dem- fi Ndufaf1 complex I (CI) de ciencies, including poor feeding, neuro- onstrated by native Western blots. Furthermore, the degeneration, and muscle weakness (17), including the eyelid gene, encoding a complex I assembly factor, was genetically map- muscles (OMIM 252010). In the late stage of disease, the anx/anx ped to the anx interval and found to be down-regulated in anx/ mice keep their eyes partly shut, possibly because of weakening anx mice. These results suggest that the anorexia and hypotha- of the eyelid muscles. Taking these data together, we hypothe- lamic neurodegeneration of the anx/anx mouse are associated sized that the starvation and neurodegeneration observed in the with dysfunction of mitochondrial complex I. anx/anx mouse could be related to defects in the mitochondrial OXPHOS (17) and possibly CI deficiency. neuropeptides | reactive oxygen species | agouti-gene related protein | In the present study, using Affymetrix microarray, we found food intake | neuroinflammation 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 he anorectic anx/anx mouse (1) is an attractive model for dis- and CI in anx/anx mice by high-resolution respirometry. The Tturbed feeding behavior, as it exhibits phenotypes associated levels of fully and partly assembled CI were studied using native with failure to thrive in infants and young children (2), anorexia Western blots, and markers for reactive oxygen species (ROS) nervosa (3), and cachexia (4). This mouse arose by a spontaneous and oxidative stress were analyzed by histochemical methods. mutation (anorexia, allele symbol anx) and is characterized by Mapping of the interval of the anx gene and mutation revealed poor appetite and reduced stomach content, and dies (likely be- that one of the CI assembly factors, the NADH dehydrogenase cause of the severe starvation) around 3 wk after birth (1). The anx/ (ubiquinone) 1α-subcomplex, assembly factor 1 (Ndufaf1)-gene, anx mice eat significantly less than their wild-type littermates, and is located in the interval. This gene was also found to be down- by postnatal day (P) 21 they weigh half as much, rendering them an regulated in anx/anx mice, specific for the anx-allele. emaciated appearance (1). These mice also exhibit a number of neurological abnormalities, such as body tremors, head weaving, Results hyperactivity, and uncoordinated gait (1). fi anx/anx – Mitochondrial CI De ciency in the Mouse Hypothalamus. Several neurotransmitter (5, 6) and neuropeptidergic (7 11) A microarray analysis of Arc from anx/anx (n = 3) and +/+ systems involved in the regulation of food intake and energy (wild-type) (n = 3) mice revealed 132 up-regulated genes and 73 metabolism are disturbed in the anx/anx mouse. The majority of anx/anx ≥ fi down-regulated genes in mice, with 1.4-fold average these ndings are centered around the hypothalamus, the origin increase or decrease (Table S1). Through an Ingenuity Pathway of a neuronal network important for the control of initiation and termination of food intake, as well as diet-induced thermo- genesis and energy expenditure. The arcuate nucleus of hypo- thalamus (Arc), situated at the interface between the periphery Author contributions: C.L., I.A.K.N., P.M.G.-R., M.K., T.J.E., T.G.M.H., M.S., and J.E.J. de- and brain, receives signals about energy status from the periph- signed research; C.L., I.A.K.N., P.M.G.-R., A.R.Z., M.K., L.R.D., D.C.R., J.M., M.T.D., M.S., and J.E.J. performed research; M.U. contributed new reagents/analytic tools; C.L., I.A.K.N., ery, such as circulating leptin and insulin levels, resulting in an- P.M.G.-R., and J.E.J. analyzed data; and C.L., I.A.K.N., P.M.G.-R., T.G.M.H., M.S., and J.E.J. orexigenic or orexigenic behavior. Two main populations of food wrote the paper. intake-regulating neurons reside in the Arc, both expressing The authors declare no conflict of interest. leptin and insulin receptors (12). One group coexpresses the two orexigenic neuropeptides neuropeptide Y (NPY) and agouti- Freely available online through the PNAS open access option. gene related protein (AGRP) (11, 13), whereas the other popula- 1C.L. and I.A.K.N. contributed equally to this work. tion expresses anorexigenic proopiomelanocortin (POMC) and 2To whom correspondence may be addressed. E-mail: [email protected] or ida.nilsson@ cocaine- and amphetamine-regulated transcript (CART) (14). ki.se. Both these groups undergo degeneration in the anorexic anx/anx This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mouse (15). By P21, anx/anx mice exhibit fewer hypothalamic 1073/pnas.1114863108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1114863108 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 Analysis (IPA) of these genes, we identified the top canonical assembly pattern of the different mitochondrial complexes in pathways most likely to be involved in the phenotype of the anx/anx anx/anx hypothalamus. This analysis showed lower levels of fully mice. The pathway analysis identified several genes related to assembled CI in the anx/anx hypothalamus compared with +/+ OXPHOS pathways and mitochondrial functions (Fig. S1 and mice, revealed by reduced amounts of protein complexes of ∼700 Table S2). Genes related to mitochondrial function and oxidative kDa, with CI-specific activity (0.66 vs. 1.64, P = 0.04; n = 10) stress (e.g., Sod1, Prdx1, Bcl211, and Cox5B) are shown in Table 1. (Fig. 1 B and C). The levels of CIII were also decreased in anx/ In addition, the top canonical pathways identified included func- anx compared with +/+ hypothalamus (0.56 vs. 1.7, P = 0.02; tions, such as cell death and morphology, cellular growth, and n = 10) (Fig. 1B). However, the amount of CII, CIV, and CV proliferation and inflammatory responses (Table S2). were unchanged (CII: 0.95 vs. 1.2, P = 0.4; n = 10; CIV: 1.0 vs. Because both the phenotype and the microarray results suggest 1.2, P = 0.7; n = 6; CV: 0.85 vs. 1.25, P = 0.5; n = 4) (Fig. 1B). hypothalamic mitochondrial dysfunction in the anx/anx mouse, we The in-gel staining confirmed CI activity of the 700-kDa band and assessed mitochondrial respiration in mechanically permeabilized revealed lower activity of CI in the hypothalamus of anx/anx (n =4) hypothalami from anx/anx and +/+ mice (n =6–9 per genotype) compared with +/+ (n = 3) mice (9.7 vs. 14.4, P = 0.01) (Fig. 1C). using high-resolution respirometry (Fig. 1A). Oxygen flux nor- malized to hypothalamus wet weight was significantly reduced in Increased Hypothalamic ROS Production and Signs of Oxidative Stress anx/anx mice compared with +/+ littermates. The anx/anx mice in the anx/anx Mouse. CI deficiency is often accompanied by oxi- showed a 40% reduction in oxygen consumption, when mito- dative stress (18).