AIF3 splicing switch triggers neurodegeneration.

BACKGROUND: Apoptosis-inducing factor (AIF), as a mitochondrial flavoprotein, plays a fundamental role in mitochondrial bioenergetics that is critical for cell survival and also mediates caspase-independent cell death once it is released from mitochondria and translocated to the nucleus under ischemic stroke or neurodegenerative diseases. Although alternative splicing regulation of AIF has been implicated, it remains unknown which AIF splicing isoform will be induced under pathological conditions and how it impacts mitochondrial functions and neurodegeneration in adult brain. METHODS: AIF splicing induction in brain was determined by multiple approaches including 5' RACE, Sanger sequencing, splicing-specific PCR assay and bottom-up proteomic analysis. The role of AIF splicing in mitochondria and neurodegeneration was determined by its biochemical properties, cell death analysis, morphological and functional alterations and animal behavior. Three animal models, including loss-of-function harlequin model, gain-of-function AIF3 knockin model and conditional inducible AIF splicing model established using either Cre-loxp recombination or CRISPR/Cas9 techniques, were applied to explore underlying mechanisms of AIF splicing-induced neurodegeneration. RESULTS: We identified a nature splicing AIF isoform lacking exons 2 and 3 named as AIF3. AIF3 was undetectable under physiological conditions but its expression was increased in mouse and human postmortem brain after stroke. AIF3 splicing in mouse brain caused enlarged ventricles and severe neurodegeneration in the forebrain regions. These AIF3 splicing mice died 2-4 months after birth. AIF3 splicing-triggered neurodegeneration involves both mitochondrial dysfunction and AIF3 nuclear translocation. We showed that AIF3 inhibited NADH oxidase activity, ATP production, oxygen consumption, and mitochondrial biogenesis. In addition, expression of AIF3 significantly increased chromatin condensation and nuclear shrinkage leading to neuronal cell death. However, loss-of-AIF alone in harlequin or gain-of-AIF3 alone in AIF3 knockin mice did not cause robust neurodegeneration as that observed in AIF3 splicing mice. CONCLUSIONS: We identified AIF3 as a disease-inducible isoform and established AIF3 splicing mouse model. The molecular mechanism underlying AIF3 splicing-induced neurodegeneration involves mitochondrial dysfunction and AIF3 nuclear translocation resulting from the synergistic effect of loss-of-AIF and gain-of-AIF3. Our study provides a valuable tool to understand the role of AIF3 splicing in brain and a potential therapeutic target to prevent/delay the progress of neurodegenerative diseases.

Apoptosis-inducing factor deficient mice fail to develop hepatic steatosis under high fat high fructose diet or bile duct ligation.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein involved in redox signalling and programmed cell death. The role of AIF has been well recognized in diabetes and obesity. However, the aspect of AIF deficiency in the development of hepatic steatosis and liver injury is unknown. Therefore, in the current study, Harlequin (Hq mutant) mouse with markedly reduced content of AIF was investigated to explore the role of AIF on the initiation of liver injury. The wild type (WT) developed physiological and pathological features of non-alcoholic fatty liver disease (NAFLD) that were not seen in the Hq mice with AIF deficiency, when fed on high fat high fructose (HFHF) diet. Following bile duct ligation (BDL), the liver associated pathological changes were less conspicuous in Hq mice as compared to WT mice. The expression of AIF protein and apoptosis was markedly lesser as compared to their respective control in Hq mice on HFHF diet. Furthermore, the genes involved in fatty acid metabolism were also altered in the group of treated Hq mice. In conclusion, Hq mice failed to develop diet induced hepatic steatosis, suggestive of a role of AIF mediated pathway in the initiation and progression of liver inflammation. Thus, partial loss of AIF appears to be hepatoprotective. SIGNIFICANCE OF THE STUDY: AIF deficiency has multiple roles in altered pathology processes and cellular metabolism, thereby compromising the cellular homeostasis. Considering the molecular functions of AIF in other organ pathology little is known about its role in diet induced liver injury. Hence, the aim of the current study was to investigate the role of AIF deficiency in liver injury and diseases with focus on NAFLD. The study will help to deliniate the mechanisms of NAFLD using Harliquin Mice.

Apoptosis inducing factor deficiency causes retinal photoreceptor degeneration. The protective role of the redox compound methylene blue.

Dysfunction in mitochondrial oxidative phosphorylation (OXPHOS) underlies a wide spectrum of human ailments known as mitochondrial diseases. Deficiencies in complex I of the electron transport chain (ETC) contribute to 30-40% of all cases of mitochondrial diseases, and leads to eye disease including optic nerve atrophy and retinal degeneration. The mechanisms responsible for organ damage in mitochondrial defects may include energy deficit, oxidative stress, and an increase in the NADH/NAD+ redox ratio due to decreased NAD+ regeneration. Currently, there is no effective treatment to alleviate human disease induced by complex I defect. Photoreceptor cells have the highest energy demand and dependence on OXPHOS for survival, and the lowest reserve capacity indicating that they are sensitive to OXPHOS defects. We investigated the effect of mitochondrial OXPHOS deficiency on retinal photoreceptors in a model of mitochondrial complex I defect (apoptosis inducing factor, AIF-deficient mice, Harlequin mice), and tested the protective effect of a mitochondrial redox compound (methylene blue, MB) on mitochondrial and photoreceptor integrity. MB prevented the reduction in the retinal thickness and protein markers for photoreceptor outer segments, Muller and ganglion cells, and altered mitochondrial integrity and function induced by AIF deficiency. In rotenone-induced complex I deficient 661 W cells (an immortalized mouse photoreceptor cell line) MB decreased the NADH/NAD+ ratio and oxidative stress without correcting the energy deficit, and improved cell survival. MB deactivated the mitochondrial stress response pathways, the unfolding protein response and mitophagy. In conclusion, preserving mitochondrial structure and function alleviates retinal photoreceptor degeneration in mitochondrial complex I defect.

AIF loss deregulates hematopoiesis and reveals different adaptive metabolic responses in bone marrow cells and thymocytes.

Mitochondrial metabolism is a tightly regulated process that plays a central role throughout the lifespan of hematopoietic cells. Herein, we analyze the consequences of the mitochondrial oxidative phosphorylation (OXPHOS)/metabolism disorder associated with the cell-specific hematopoietic ablation of apoptosis-inducing factor (AIF). AIF-null (AIF-/Y ) mice developed pancytopenia that was associated with hypocellular bone marrow (BM) and thymus atrophy. Although myeloid cells were relatively spared, the B-cell and erythroid lineages were altered with increased frequencies of precursor B cells, pro-erythroblasts I, and basophilic erythroblasts II. T-cell populations were dramatically reduced with a thymopoiesis blockade at a double negative (DN) immature state, with DN1 accumulation and delayed DN2/DN3 and DN3/DN4 transitions. In BM cells, the OXPHOS/metabolism dysfunction provoked by the loss of AIF was counterbalanced by the augmentation of the mitochondrial biogenesis and a shift towards anaerobic glycolysis. Nevertheless, in a caspase-independent process, the resulting excess of reactive oxygen species compromised the viability of the hematopoietic stem cells (HSC) and progenitors. This led to the progressive exhaustion of the HSC pool, a reduced capacity of the BM progenitors to differentiate into colonies in methylcellulose assays, and the absence of cell-autonomous HSC repopulating potential in vivo. In contrast to BM cells, AIF-/Y thymocytes compensated for the OXPHOS breakdown by enhancing fatty acid ß-oxidation. By over-expressing CPT1, ACADL and PDK4, three key enzymes facilitating fatty acid ß-oxidation (e.g., palmitic acid assimilation), the AIF-/Y thymocytes retrieved the ATP levels of the AIF +/Y cells. As a consequence, it was possible to significantly reestablish AIF-/Y thymopoiesis in vivo by feeding the animals with a high-fat diet complemented with an antioxidant. Overall, our data reveal that the mitochondrial signals regulated by AIF are critical to hematopoietic decision-making. Emerging as a link between mitochondrial metabolism and hematopoietic cell fate, AIF-mediated OXPHOS regulation represents a target for the development of new immunomodulatory therapeutics.

Paradoxical Inhibition of Glycolysis by Pioglitazone Opposes the Mitochondriopathy Caused by AIF Deficiency.

Mice with the hypomorphic AIF-Harlequin mutation exhibit a highly heterogeneous mitochondriopathy that mostly affects respiratory chain complex I, causing a cerebral pathology that resembles that found in patients with AIF loss-of-function mutations. Here we describe that the antidiabetic drug pioglitazone (PIO) can improve the phenotype of a mouse Harlequin (Hq) subgroup, presumably due to an inhibition of glycolysis that causes an increase in blood glucose levels. This glycolysis-inhibitory PIO effect was observed in cultured astrocytes from Hq mice, as well as in human skin fibroblasts from patients with AIF mutation. Glycolysis inhibition by PIO resulted from direct competitive inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Moreover, GAPDH protein levels were reduced in the cerebellum and in the muscle from Hq mice that exhibited an improved phenotype upon PIO treatment. Altogether, our results suggest that excessive glycolysis participates to the pathogenesis of mitochondriopathies and that pharmacological inhibition of glycolysis may have beneficial effects in this condition.

Loss of apoptosis-inducing factor critically affects MIA40 function.

Mitochondrial apoptosis-inducing factor (AIF) influences the oxidative phosphorylation (OXPHOS) system and can be recruited as a mediator of cell death. Pathogenic mutations in the AIFM1 gene cause severe human diseases. Clinical manifestations include inherited peripheral neuropathies, prenatal cerebral abnormalities and progressive mitochondrial encephalomyopathies. In humans, rodents and invertebrates, AIF deficiency results in loss of respiratory complexes and, therefore, impaired OXPHOS. The molecular mechanisms underlying AIF-induced mitochondrial dysfunction remain elusive. Here we show that AIF physically interacts with the oxidoreductase CHCHD4/MIA40. In patient-derived fibroblasts as well as in tissues and glia cells from Harlequin (Hq) mutant mice, AIF deficiency correlates with decreased MIA40 protein levels, without affecting mRNA transcription. Importantly, MIA40 overexpression counteracts loss of respiratory subunits in Hq cells. Together, our findings suggest that MIA40 reduction contributes to the effects of AIF deficiency on OXPHOS, as it may impact on the correct assembly and maintenance of the respiratory subunits. This may be relevant for the development of new therapeutic approaches for AIF-related mitochondrial disorders.

The regulatory roles of apoptosis-inducing factor in the formation and regression processes of ocular neovascularization.

The role of apoptosis in the formation and regression of neovascularization is largely hypothesized, although the detailed mechanism remains unclear. Inflammatory cells and endothelial cells both participate and interact during neovascularization. During the early stage, these cells may migrate into an angiogenic site and form a pro-angiogenic microenvironment. Some angiogenic vessels appear to regress, whereas some vessels mature and remain. The control mechanisms of these processes, however, remain unknown. Previously, we reported that the prevention of mitochondrial apoptosis contributed to cellular survival via the prevention of the release of proapoptotic factors, such as apoptosis-inducing factor (AIF) and cytochrome c. In this study, we investigated the regulatory role of cellular apoptosis in angiogenesis using two models of ocular neovascularization: laser injury choroidal neovascularization and VEGF-induced corneal neovascularization in AIF-deficient mice. Averting apoptosis in AIF-deficient mice decreased apoptosis of leukocytes and endothelial cells compared to wild-type mice and resulted in the persistence of these cells at angiogenic sites in vitro and in vivo. Consequently, AIF deficiency expanded neovascularization and diminished vessel regression in these two models. We also observed that peritoneal macrophages from AIF-deficient mice showed anti-apoptotic survival compared to wild-type mice under conditions of starvation. Our data suggest that AIF-related apoptosis plays an important role in neovascularization and that mitochondria-regulated apoptosis could offer a new target for the treatment of pathological angiogenesis.

Vision deficits precede structural losses in a mouse model of mitochondrial dysfunction and progressive retinal degeneration.

Current animal models of retinal disease often involve the rapid development of a retinal disease phenotype; however, this is at odds with age-related diseases that take many years to manifest clinical symptoms. The present study was performed to examine an apoptosis-inducing factor (Aif)-deficient model, the harlequin carrier mouse (X(hq)X), and determine how mitochondrial dysfunction and subsequent accelerated aging affect the function and structure of the mouse retina. Vision and eye structure for cohorts of 6 X(hq)X and 6 wild type mice at 3, 11, and 15 months of age were studied using in vivo electroretinography (ERG), and optical coherence tomography (OCT). Retinal superoxide levels were determined in situ using dihydroethidium (DHE) histochemistry. Retinal cell counts were quantified post mortem using hematoxylin and eosin (H&E) staining. ERG analysis of X(hq)X retinal function indicated a reduction in b-wave amplitude significant at 3 months of age (p < 0.05), declining further with age. However, retinal neuron counts demonstrated the absence of physical degeneration at 3 and 11 months of age despite significant reduction in ERG b-wave amplitude. Superoxide anion levels were elevated in the ganglion cell, inner nuclear and outer nuclear layers of the retina (p < 0.01, p < 0.01, and p < 0.001, respectively) of 11-month-old X(hq)X mice in comparison to wild type, preceding the structural losses observed at 15 mos. Early onset of retinal function deficits occurred independently of neuron loss. Changes in neurotransmitter localization in the stressed retina may account for the early and significant reduction in retinal function. This remodeling of retinal neurochemistry in response to stress may be a relevant mechanism in the progression of normal retinal aging and early stages of some retinal degenerative diseases.

Role of apoptosis inducing factor (AIF) for hippocampal neuronal cell death following global cerebral ischemia in mice.

The molecular mechanisms of neuronal cell death following circulatory arrest are still not fully understood. In the current study we investigated the role of apoptosis-inducing factor (AIF), a major caspase-independent mitochondrial cell death protein, for neuronal cell death following global cerebral ischemia (GCI). C57/Bl6 or low AIF expressing Harlequin mutant mice (AIF(low)) and their wild-type littermates were subjected to 10 min of GCI. DNA damage, nuclear pathology, and localization of AIF were investigated 6, 24, and 72 h after GCI by TUNEL and DAPI staining, and immunohistochemistry, respectively. Cell death of hippocampal CA1 neurons following GCI was associated with nuclear translocation of AIF, nuclear pyknosis, and DNA fragmentation, i.e. ∼80% of all TUNEL-positive neurons had nuclear AIF staining. In AIF(low) mice neuronal cell loss was reduced by 60% (p<0.02). The current experiments suggest that AIF-mediated signaling represents a novel mechanism of neuronal cell death following GCI.

Apoptosis inducing factor deficiency causes reduced mitofusion 1 expression and patterned Purkinje cell degeneration.

Alteration in mitochondrial dynamics has been implicated in many neurodegenerative diseases. Mitochondrial apoptosis inducing factor (AIF) plays a key role in multiple cellular and disease processes. Using immunoblotting and flow cytometry analysis with Harlequin mutant mice that have a proviral insertion in the AIF gene, we first revealed that mitofusion 1 (Mfn1), a key mitochondrial fusion protein, is significantly diminished in Purkinje cells of the Harlequin cerebellum. Next, we investigated the cerebellar pathology of Harlequin mice in an age-dependent fashion, and identified a striking process of progressive and patterned Purkinje cell degeneration. Using immunohistochemistry with zebrin II, the most studied compartmentalization marker in the cerebellum, we found that zebrin II-negative Purkinje cells first started to degenerate at 7 months of age. By 11 months of age, almost half of the Purkinje cells were degenerated. Subsequently, most of the Purkinje cells disappeared in the Harlequin cerebellum. The surviving Purkinje cells were concentrated in cerebellar lobules IX and X, where these cells were positive for heat shock protein 25 and resistant to degeneration. We further showed that the patterned Purkinje cell degeneration was dependent on caspase but not poly(ADP-ribose) polymerase-1 (PARP-1) activation, and confirmed the marked decrease of Mfn1 in the Harlequin cerebellum. Our results identified a previously unrecognized role of AIF in Purkinje cell degeneration, and revealed that AIF deficiency leads to altered mitochondrial fusion and caspase-dependent cerebellar Purkinje cell loss in Harlequin mice. This study is the first to link AIF and mitochondrial fusion, both of which might play important roles in neurodegeneration.

Apoptosis-inducing factor deficiency sensitizes dopaminergic neurons to parkinsonian neurotoxins.

OBJECTIVE: Mitochondrial complex I deficits have long been associated with Parkinson disease (PD). However, it remains unknown whether such defects represent a primary event in dopaminergic neurodegeneration. METHODS: Apoptosis-inducing factor (AIF) is a mitochondrial protein that, independently of its proapoptotic properties, plays an essential physiologic role in maintaining a fully functional complex I. We used AIF-deficient harlequin (Hq) mice, which exhibit structural deficits in assembled complex I, to determine whether primary complex I defects linked to AIF depletion may cause dopaminergic neurodegeneration. RESULTS: Despite marked reductions in mitochondrial complex I protein levels, Hq mice did not display apparent alterations in the dopaminergic nigrostriatal system. However, these animals were much more susceptible to exogenous parkinsonian complex I inhibitors, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Subtoxic doses of MPTP, unable to cause damage to wild-type animals, produced marked nigrostriatal dopaminergic degeneration in Hq mice. This effect was associated with exacerbated complex I inhibition and increased production of mitochondrial-derived reactive oxygen species (ROS) in Hq brain mitochondria. The antioxidant superoxide dismutase-mimetic compound tempol was able to reverse the increased susceptibility of Hq mice to MPTP. Supporting an instrumental role for mitochondrial-derived ROS in PD-related neurodegeneration, transgenic mice overexpressing mitochondrially targeted catalase exhibited an attenuation of MPTP-induced mitochondrial ROS and dopaminergic cell death. INTERPRETATION: Structural complex I alterations linked to AIF deficiency do not cause dopaminergic neurodegeneration but increase the susceptibility of dopaminergic neurons to exogenous parkinsonian neurotoxins, reinforcing the concept that genetic and environmental factors may interact in a common molecular pathway to trigger PD.

Apoptose e expressão de Bcl-2 e das caspases 3 e 8 em placenta bovina, em diferentes estádios de gestação / Apoptosis and expression of Bcl-2 and caspases 3 and 8 in placenta of cows at different pregnancy stages

Apoptose e seus mecanismos reguladores são eventos fisiológicos cruciais para a manutenção da homeostase placentária, e o desequilíbrio desses processos pode comprometer a função placentária e, consequentemente, o sucesso da gravidez. Neste estudo, investigou-se a apoptose utilizando-se histomorfometria em lâminas coradas em HE e submetidas à reação de TUNEL. Além disso, avaliou-se a expressão de Bcl-2 e das caspases 8 e 3, pela reação de polimerase em cadeia em tempo real, em placentas saudáveis em diferentes estádios de gestação. Amostras de placentônios de vacas com quatro, seis e nove meses de gestação foram colhidas e processadas. O índice apoptótico aumentou progressivamente com o avanço da gestação. Tanto o Bcl-2 quanto as caspases 3 e 8 foram expressas nos três períodos estudados, sendo a expressão de Bcl-2 menor que a de caspase 8, que é menor que a de caspase 3. Estes resultados indicam que essas moléculas estão envolvidas na via apoptótica ativada na maturação placentária, exibindo um padrão de expressão ao longo da gestação e contribuindo para o equilíbrio fisiológico da celularidade e renovação celular na placenta bovina.

Apoptosis and its regulating mechanisms are crucial physiological events for the maintenance of the placental homostasis; and disequilibrium of these processes may compromise placental function and the success of the pregnancy. In this study, apoptosis was investigated by histomorphometry using slides stained with HE and TUNEL reaction. Besides that, Bcl-2 and caspases 8 and 3 expression were evaluated by real time polymerase chain reaction in healthy placentas under different gestacional ages. Samples of placentones of cows at 4th, 6th, and 9th months of gestation were harvested and processed. The apoptotic index gradually increased with the advance of the gestation. Bcl-2 and caspases 3 and 8 were expressed in all the studied periods, being the expression of Bcl-2 lower than that of caspase 8, which was lower than caspase 3. These results indicate that these molecules are involved in the activated apoptotic way in the placental maturation, showing a standard expression throughout the gestation and contributing for the physiological balance of the cellularity and cellular turn over in bovine placenta.

Apoptosis-inducing factor deficiency decreases the proliferation rate and protects the subventricular zone against ionizing radiation.

Cranial radiotherapy in children often leads to progressive cognitive decline. We have established a rodent model of irradiation-induced injury to the young brain. A single dose of 8 Gy was administered to the left hemisphere of postnatal day 10 (P10) mice. Harlequin (Hq) mice, carrying the hypomorphic apoptosis-inducing factor AIF(Hq) mutation, express 60% less AIF at P10 and displayed significantly fewer dying cells in the subventricular zone (SVZ) 6 h after IR, compared with wild type (Wt) littermates. Irradiated cyclophilin A-deficient (CypA(-/-)) mice confirmed that CypA has an essential role in AIF-induced apoptosis after IR. Hq mice displayed no reduction in SVZ size 7 days after IR, whereas 48% of the SVZ was lost in Wt mice. The proliferation rate was lower in the SVZ of Hq mice. Cultured neural precursor cells from the SVZ of Hq mice displayed a slower proliferation rate and were more resistant to IR. IR preferentially kills proliferating cells, and the slower proliferation rate in the SVZ of Hq mice may, at least partly, explain the protective effect of the Hq mutation. Together, these results indicate that targeting AIF may provide a fruitful strategy for protection of normal brain tissue against the detrimental side effects of IR.

Deletion of the mitochondrial flavoprotein apoptosis inducing factor (AIF) induces beta-cell apoptosis and impairs beta-cell mass.

BACKGROUND: Apoptosis is a hallmark of beta-cell death in both type 1 and type 2 diabetes mellitus. Understanding how apoptosis contributes to beta-cell turnover may lead to strategies to prevent progression of diabetes. A key mediator of apoptosis, mitochondrial function, and cell survival is apoptosis inducing factor (AIF). In the present study, we investigated the role of AIF on beta-cell mass and survival using the Harlequin (Hq) mutant mice, which are hypomorphic for AIF. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemical evaluation of pancreata from Hq mutant mice displayed much smaller islets compared to wild-type mice (WT). Analysis of beta-cell mass in these mice revealed a greater than 4-fold reduction in beta-cell mass together with an 8-fold increase in beta-cell apoptosis. Analysis of cell cycle dynamics, using BrdU pulse as a marker for cells in S-phase, did not detect significant differences in the frequency of beta-cells in S-phase. In contrast, double staining for phosphorylated Histone H3 and insulin showed a 3-fold increase in beta-cells in the G2 phase in Hq mutant mice, but no differences in M-phase compared to WT mice. This suggests that the beta-cells from Hq mutant mice are arrested in the G2 phase and are unlikely to complete the cell cycle. beta-cells from Hq mutant mice display increased sensitivity to hydrogen peroxide-induced apoptosis, which was confirmed in human islets in which AIF was depleted by siRNA. AIF deficiency had no effect on glucose stimulated insulin secretion, but the impaired effect of hydrogen peroxide on beta-cell function was potentiated. CONCLUSIONS/SIGNIFICANCE: Our results indicate that AIF is essential for maintaining beta-cell mass and for oxidative stress response. A decrease in the oxidative phosphorylation capacity may counteract the development of diabetes, despite its deleterious effects on beta-cell survival.

Loss of apoptosis-inducing factor results in cell-type-specific neurogenesis defects.

Mitochondrial dysfunction is commonly associated with neurodegeneration in the aging brain. In addition, the importance of mitochondrial function during brain development is illustrated by the neurological deficits observed in infants with mitochondrial complex deficiencies. However, the extent to which abnormalities in mitochondrial function might impact neurogenesis during brain development is not well understood. Previously, we demonstrated that adult harlequin (Hq) mutant mice, which have an 80% reduction in the mitochondrial protein apoptosis-inducing factor (AIF), exhibited signs of oxidative stress and progressive loss of adult cerebellar and retinal neurons. To assess whether in addition to its role in postmitotic neuron survival Aif is also necessary for cerebellar development, we analyzed embryos in which Aif was deleted in the prospective midbrain and cerebellum at a very early stage of development using an En1 (engrailed 1) promoter-driven cre recombinase gene. These mutant mice, which died at birth, had midbrain defects and dramatic deficits in cerebellar Purkinje and granule cell precursors. Additional analysis revealed that Aif-null Purkinje cell precursors prematurely entered S-phase, but most failed to undergo mitosis and ultimately died via apoptosis. In contrast, proliferation of mutant granule cell precursors was blocked before S-phase. Mice in which Aif was deleted later in embryogenesis using a nestin promoter-driven cre gene survive for several days after birth, and postnatal granule cell precursors in these mice also failed to enter S-phase. Our results indicate that the loss of Aif results in cell cycle abnormalities in a neuron-specific manner during cerebellar development.

Is energy deficiency good in moderation?

In this issue, Pospisilik et al. (2007) demonstrate that a reduction in mitochondrial oxidative phosphorylation protects mice against obesity and diabetes. This finding suggests that the moderate deficiency in oxidative phosphorylation that is observed in peripheral tissues of insulin-resistant humans is not a causative factor in diabetes but may instead be a compensatory response.

Targeted deletion of AIF decreases mitochondrial oxidative phosphorylation and protects from obesity and diabetes.

Type-2 diabetes results from the development of insulin resistance and a concomitant impairment of insulin secretion. Recent studies place altered mitochondrial oxidative phosphorylation (OxPhos) as an underlying genetic element of insulin resistance. However, the causative or compensatory nature of these OxPhos changes has yet to be proven. Here, we show that muscle- and liver-specific AIF ablation in mice initiates a pattern of OxPhos deficiency closely mimicking that of human insulin resistance, and contrary to current expectations, results in increased glucose tolerance, reduced fat mass, and increased insulin sensitivity. These results are maintained upon high-fat feeding and in both genetic mosaic and ubiquitous OxPhos-deficient mutants. Importantly, the effects of AIF on glucose metabolism are acutely inducible and reversible. These findings establish that tissue-specific as well as global OxPhos defects in mice can counteract the development of insulin resistance, diabetes, and obesity.

Apoptosis-inducing factor deficiency induces early mitochondrial degeneration in brain followed by progressive multifocal neuropathology.

Apoptosis-inducing factor (AIF) deficiency compromises oxidative phosphorylation. Harlequin mice, in which AIF is downregulated, develop a severe mitochondrial complex I (CI) deficiency, suggesting that Harlequin mice may represent a natural model of the most common oxidative phosphorylation disorders. However, the brain phenotype specifically involves the cerebellum, whereas human CI deficiencies often manifest as complex multifocal neuropathologies. To evaluate whether this model can be used as to study CI-deficient disorders, the whole brain of Harlequin mice was investigated during the course of the disease. Neurodegeneration was not restricted to the cerebellum but progressively affected thalamic, striatal, and cortical regions as well. Strong astroglial and microglial activation with extensive vascular proliferation was observed by 4 months of age in thalamic, striatal, and cerebellar nuclei associated with somatosensory-motor pathways. At 2 months of age, degenerating mitochondria were observed in most cells in these structures, even in nondegenerating neurons, a finding that indicates mitochondrial injury is a cause rather than an effect of neuronal cell death. Thus, apoptosis-inducing factor deficiency induces early mitochondrial degeneration, followed by progressive multifocal neuropathology (a phenotype broader than previously described), and resembles some histopathologic features of devastating human neurodegenerative mitochondriopathies associated with CI deficiency.

Apoptosis-inducing factor is a major contributor to neuronal loss induced by neonatal cerebral hypoxia-ischemia.

Nine-day-old harlequin (Hq) mice carrying the hypomorphic apoptosis-inducing factor (AIF)(Hq) mutation expressed 60% less AIF, 18% less respiratory chain complex I and 30% less catalase than their wild-type (Wt) littermates. Compared with Wt, the infarct volume after hypoxia-ischemia (HI) was reduced by 53 and 43% in male (YX(Hq)) and female (X(Hq)X(Hq)) mice, respectively (P<0.001). The Hq mutation did not inhibit HI-induced mitochondrial release of cytochrome c or activation of calpain and caspase-3. The broad-spectrum caspase inhibitor quinoline-Val-Asp(OMe)-CH(2)-PH (Q-VD-OPh) decreased the activation of all detectable caspases after HI, both in Wt and Hq mice. Q-VD-OPh reduced the infarct volume equally in Hq and in Wt mice, and the combination of Hq mutation and Q-VD-OPh treatment showed an additive neuroprotective effect. Oxidative stress leading to nitrosylation and lipid peroxidation was more pronounced in ischemic brain areas from Hq than Wt mice. The antioxidant edaravone decreased oxidative stress in damaged brains, more pronounced in the Hq mice, and further reduced brain injury in Hq but not in Wt mice. Thus, two distinct strategies can enhance the neuroprotection conferred by the Hq mutation, antioxidants, presumably compensating for a defect in AIF-dependent redox detoxification, and caspase inhibitors, presumably interrupting a parallel pathway leading to cellular demise.