Interventions to improve mitochondrial function in a mouse model of GRACILE syndrome, a complex III disorder

A rare homozygous BCS1Lc.A232G (Ser78Gly, p.S78G) mutation in infants causes GRACILE syndrome, which is a severe mitochondrial respiratory chain complex III (CIII) disorder resulting in multiple organ dysfunction and early lethality. Pathogenesis mechanisms have been studied using our viable Bcs1lp.S78G knock-in mouse model. The mouse model replicates most clinical phenotypes, such as growth restriction, hepatopathy, and tubulopathy. Like patients, the survival of homozygous mice is reduced (to 35-45 days, P35-P45 in the C57BL/6JBomTac background), mainly because of severe hypoglycemia. Aiming to improve the glycemic balance we performed an intervention with a high sugar (60% dextrose) diet. This diet did not improve energy metabolism and resulted in slightly decreased survival despite apparent normalization of some plasma metabolites. For subsequent studies, we bred the Bcs1lc.A232G mutation into a C57BL/6JCrl background, in which the survival was five-fold longer (approximately 200 days). Moreover, the extended survival brought novel phenotypes, such as encephalopathy and late-onset cardiomyopathy. In this genetic background, we investigated the effect of ketogenic diet on disease progression. The ketogenic diet had a beneficial impact on liver disease, but it had adverse effects upon long-term feeding, resulting in shortened survival. In the third study, we introduced an alternative oxidase (AOX) transgene into the Bcs1lp.S78G mice to improve respiratory chain function. The ubiquitous expression of AOX, which should bypass electron transfer and relieve CIII blockade, prevented lethal cardiomyopathy and renal-tubular atrophy, and delayed focal astrogliosis in the somatosensory cortex of the brain. The beneficial effects of AOX extended the median survival of the homozygotes to median P590. The main conclusions from these studies are that the Bcs1lp.S78G mice in a C57BL/6JCrl background present with both the known early-onset manifestations of GRACILE syndrome and some later onset manifestations found in other CIII deficiencies. The dietary interventions had limited benefits, probably because of a severe course of the disease. In contrast, bypassing the blocked electron flow using AOX had a robust beneficial effect, mainly in tissues or cells with high ATP demand such as the heart and renal proximal tubular cells.GRACILE-oireyhtymä on suomalaiseen tautiperimään kuuluva vaikea vastasyntyneiden mitokondriosairaus, jonka aiheuttaa homotsygoottinen pistemutaatio (c.A232G, p.Ser78Gly) BCS1L-geenissä. GRACILE-oireyhtymässä mitokondrioiden hengitysketjun kompleksi III (CIII) on viallinen, mikä johtaa vaikeaan aineenvaihdunnan häiriöön, maksa- ja munuaissairauteen ja varhaiseen kuolemaan. Sairauden syntymekanismien ja hoitomahdollisuuksien tutkimuksessa on osoittautunut erittäin hyödylliseksi hiirimalli, jossa potilaiden pistemutaatio on viety hiiren genomiin. Hiirimallissa toistuvat useimmat potilaiden oireet, kuten kasvuhäiriö, alhainen verensokeri ja maksa- ja munuaissairaus. Homotsygoottiset hiiret (Bcs1lp.S78G) kuolevat C57BL/6JBomTac-kannassa vain 30-40 päivän ikäisinä, todennäköisesti hypoglykemiaan. Sen vuoksi tutkimme tässä väitöskirjassa sokeripitoisen ruoan (60 % glukoosia) vaikutusta hiirten verensokeriin ja elinikään. Sokerilisä johti kuitenkin jopa hiukan lyhempään elinikään huolimatta siitä, että se näytti korjaavan joidenkin aineenvaiduntatuotteiden pitoisuuksia plasmassa. Seuraavissa osatöissä huomasimme, että C57BL/6JCrl-kannassa hiiret elivät viisi kertaa pidempään (noin 200 päivää) kuin aikaisemmin Lundin yliopistossa käytetyssä kannassa, ja niille kehittyi uusia myöhään alkavia oireita kuten vaikea sydänlihassairaus ja aivomuutoksia. Tässä kannassa tutkimme ensin rasvapitoisen (ketogeenisen) ruokavalion vaikutusta maksasairauden etenemiseen. Ketogeenisella ruokavaliolla oli selvä myönteinen vaikutus maksasairaukseen ensimmäisten kolmen kuukauden aikana, mutta pitkäkestoinen ruokinta aiheutti haittavaikutuksia. Kolmannessa osatutkimuksessa risteytimme Bcs1l-mutaatiota kantavia hiiriä vaihtoehtoista oksidaasia (AOX) ilmentävän siirtogeenisen hiirilinjan kanssa. AOX voi parantaa hengitysketjun alkuosan toimintaa ohjaamalla esimerkiksi CIII:n toimintahäiriön vuoksi pysähtynyttä elektronien kulkua koentsyymi Q:lta suoraan hapelle. AOX:n esti kokonaan sydänlihassairauden ja munuaissurkastuman kehittymisen Bcs1lp.S78G-hiirille ja hidasti aivomuutosten etenemistä. Sydänsairauden estymisen johdosta AOX-siirtogeeniä kantavien mutanttihiirten keskimääräinen elinikä piteni 210 päivästä 590:een. Väitöskirjatutkimusten tärkeimpiä johtopäätöksiä ovat, että C57BL/6JCrl-kannassa Bcs1lp.S78G-hiirillä ilmenee sekä GRACILE-oireyhtymälle tyypillisiä alkuvaiheen oireita, kuten kasvuhäiriö ja maksasairaus, että myöhemmin kehittyviä uusia oireita, kuten sydänlihassairaus, joita esiintyy muissa CIII-puutossairauksissa. Ruokavaliohoidoilla oli edullista vaikutusta maksasairauteen, mutta muutoin vaikutukset olivat vähäisiä tässä vaikean mitokondriosairauden mallissa. Sitä vastoin hengitysketjun pysähtyneen elektronivirtauksen palauttaminen AOXentsyymin avulla oli ennennäkemättömän tehokas keino hidastaa tai jopa estää pysyvästi vakavia oireita, ja vaikutus oli selvin kudoksissa tai soluissa, joilla on suuri energiankulutus, kuten sydämessä ja munuaisten tubuluksissa

The Limits of Lawyering: Legal Opinions in Structured Finance

Significant controversy surrounds the issuance of legal opinions in structured finance transactions, particularly where accountants separately use these opinions, beyond their traditional primary use, for determining whether to characterize the transactions as debt. Reflecting at its core the unresolved boundaries between public and private in financial transactions, this controversy raises important issues of first impression: To what extent, for example, should lawyers be able to issue legal opinions that create negative externalities? Furthermore, what should differentiate the roles of lawyers and accountants in disclosing information to investors? Resolution of these issues not only helps to demystify the mystique, and untangle the morass, of legal-opinion giving but also affects the very viability of the securitization industry, which dominates American, and increasingly global, financing

Inter-organellar and systemic responses to impaired mitochondrial matrix protein import in skeletal muscle

Effective protein import from cytosol is critical for mitochondrial functions and metabolic regulation. We describe here the mammalian muscle-specific and systemic consequences to disrupted mitochondrial matrix protein import by targeted deletion of the mitochondrial HSP70 co-chaperone GRPEL1. Muscle-specific loss of GRPEL1 caused rapid muscle atrophy, accompanied by shut down of oxidative phosphorylation and mitochondrial fatty acid oxidation, and excessive triggering of proteotoxic stress responses. Transcriptome analysis identified new responders to mitochondrial protein import toxicity, such as the neurological disease-linked intermembrane space protein CHCHD10. Besides communication with ER and nucleus, we identified crosstalk of distressed mitochondria with peroxisomes, in particular the induction of peroxisomal Acyl-CoA oxidase 2 (ACOX2), which we propose as an ATF4-regulated peroxisomal marker of integrated stress response. Metabolic profiling indicated fatty acid enrichment in muscle, a shift in TCA cycle intermediates in serum and muscle, and dysregulated bile acids. Our results demonstrate the fundamental importance of GRPEL1 and provide a robust model for detecting mammalian inter-organellar and systemic responses to impaired mitochondrial matrix protein import and folding.Peer reviewe

Effect of High-Carbohydrate Diet on Plasma Metabolome in Mice with Mitochondrial Respiratory Chain Complex III Deficiency

Mitochondrial disorders cause energy failure and metabolic derangements. Metabolome profiling in patients and animal models may identify affected metabolic pathways and reveal new biomarkers of disease progression. Using liver metabolomics we have shown a starvation-like condition in a knock-in (Bcs1l(c.232A>G)) mouse model of GRACILE syndrome, a neonatal lethal respiratory chain complex III dysfunction with hepatopathy. Here, we hypothesized that a high-carbohydrate diet (HCD, 60% dextrose) will alleviate the hypoglycemia and promote survival of the sick mice. However, when fed HCD the homozygotes had shorter survival (mean +/- SD, 29 +/- 2.5 days, n = 21) than those on standard diet (33 +/- 3.8 days, n = 30), and no improvement in hypoglycemia or liver glycogen depletion. We investigated the plasma metabolome of the HCD- and control diet-fed mice and found that several amino acids and urea cycle intermediates were increased, and arginine, carnitines, succinate, and purine catabolites decreased in the homozygotes. Despite reduced survival the increase in aromatic amino acids, an indicator of liver mitochondrial dysfunction, was normalized on HCD. Quantitative enrichment analysis revealed that glycine, serine and threonine metabolism, phenylalanine and tyrosine metabolism, and urea cycle were also partly normalized on HCD. This dietary intervention revealed an unexpected adverse effect of high-glucose diet in complex III deficiency, and suggests that plasma metabolomics is a valuable tool in evaluation of therapies in mitochondrial disorders.Peer reviewe

A spontaneous mitonuclear epistasis converging on Rieske Fe-S protein exacerbates complex III deficiency in mice

We previously observed an unexpected fivefold (35 vs. 200 days) difference in the survival of respiratory chain complex III (CIII) deficient Bcs1/(p.S78G) mice between two congenic backgrounds. Here, we identify a spontaneous homoplasmic mtDNA variant (m.G14904A, mt-Cyb(p.D254N)), affecting the CIII subunit cytochrome b (MT-CYB), in the background with short survival. We utilize maternal inheritance of mtDNA to confirm this as the causative variant and show that it further decreases the low CIII activity in Bcs1/(p.S78G) tissues to below survival threshold by 35 days of age. Molecular dynamics simulations predict D254N to restrict the flexibility of MT-CYB ef loop, potentially affecting RISP dynamics. In Rhodobacter cytochrome bc(1) complex the equivalent substitution causes a kinetics defect with longer occupancy of RISP head domain towards the quinol oxidation site. These findings represent a unique case of spontaneous mitonuclear epistasis and highlight the role of mtDNA variation as modifier of mitochondrial disease phenotypes.Peer reviewe

Ketogenic diet attenuates hepatopathy in mouse model of respiratory chain complex III deficiency caused by a Bcs1l mutation

Mitochondrial disorders are among the most prevalent inborn errors of metabolism but largely lack treatments and have poor outcomes. High-fat, low-carbohydrate ketogenic diets (KDs) have shown beneficial effects in mouse models of mitochondrial myopathies, with induction of mitochondrial biogenesis as the suggested main mechanism. We fed KD to mice with respiratory chain complex III (CIII) deficiency and progressive hepatopathy due to mutated BCS1L, a CIII assembly factor. The mutant mice became persistently ketotic and tolerated the KD for up to 11 weeks. Liver disease progression was attenuated by KD as shown by delayed fibrosis, reduced cell death, inhibition of hepatic progenitor cell response and stellate cell activation, and normalization of liver enzyme activities. Despite no clear signs of increased mitochondrial biogenesis in the liver, CIII assembly and activity were improved and mitochondrial morphology in hepatocytes normalized. Induction of hepatic glutathione transferase genes and elevated total glutathione level were normalized by KD. Histological findings and transcriptome changes indicated modulation of liver macrophage populations by the mutation and the diet. These results reveal a striking beneficial hepatic response to KD in mice with mitochondrial hepatopathy and warrant further investigations of dietary modification in the management of these conditions in patients.Peer reviewe

Alternative oxidase-mediated respiration prevents lethal mitochondrial cardiomyopathy

Alternative oxidase (AOX) is a non-mammalian enzyme that can bypass blockade of the complex III-IV segment of the respiratory chain (RC). We crossed a Ciona intestinalis AOX transgene into RC complex III (cIII)-deficient Bcs1l(p.S78G) knock-in mice, displaying multiple visceral manifestations and premature death. The homozygotes expressing AOX were viable, and their median survival was extended from 210 to 590 days due to permanent prevention of lethal cardiomyopathy. AOX also prevented renal tubular atrophy and cerebral astrogliosis, but not liver disease, growth restriction, or lipodystrophy, suggesting distinct tissue-specific pathogenetic mechanisms. Assessment of reactive oxygen species (ROS) production and damage suggested that ROS were not instrumental in the rescue. Cardiac mitochondrial ultrastructure, mitochondrial respiration, and pathological transcriptome and metabolome alterations were essentially normalized by AOX, showing that the restored electron flow upstream of cIII was sufficient to prevent cardiac energetic crisis and detrimental decompensation. These findings demonstrate the value of AOX, both as a mechanistic tool and a potential therapeutic strategy, for cIII deficiencies.Peer reviewe

NAD+ repletion produces no therapeutic effect in mice with respiratory chain complex III deficiency and chronic energy deprivation

Biosynthetic precursors of NAD+ can replenish a decreased cellular NAD+ pool and, supposedly via sirtuin (SIRT) deacetylases, improvemitochondrial function.Wefound decreased hepaticNAD+ concentration and downregulated biosynthesis in Bcs1lp.S78G knock-in mice with respiratory chain complex III deficiency and mitochondrial hepatopathy. Aiming at ameliorating disease progression via NAD+ repletion and improved mitochondrial function, we fed thesemice nicotinamide riboside (NR), aNAD+ precursor. A targetedmetabolomics verified successful administration and suggested enhancedNAD+ biosynthesis in the treated mice, although hepaticNAD+ concentrationwas unchanged at the end point. In contrast to our expectations,NRdid not improve the hepatopathy, hepatic mitochondrial respiration, or survival of Bcs1lp.S78G mice. We linked this lack of therapeutic effect to NAD+-independent activation of SIRT-1 and -3 via AMPK and cAMP signaling related to the starvation-like metabolic state of Bcs1lp.S78G mice. In summary, we describe an unusual metabolic state with NAD+ depletion accompanied by energy deprivation signals, uncompromised SIRT function, and upregulated oxidative metabolism. Our study highlights that the knowledge of the underlying complexmetabolic alterations is criticalwhen designing therapies formitochondrial dysfunction

Obesity-linked homologues TfAP-2 and Twz establish meal frequency in Drosophila melanogaster

In all animals managing the size of individual meals and frequency of feeding is crucial for metabolic homeostasis. In the current study we demonstrate that the noradrenalin analogue octopamine and the cholecystokinin (CCK) homologue Drosulfakinin (Dsk) function downstream of TfAP-2 and Tiwaz (Twz) to control the number of meals in adult flies. Loss of TfAP-2 or Twz in octopaminergic neurons increased the size of individual meals, while overexpression of TfAP-2 significantly decreased meal size and increased feeding frequency. Of note, our study reveals that TfAP-2 and Twz regulate octopamine signaling to initiate feeding; then octopamine, in a negative feedback loop, induces expression of Dsk to inhibit consummatory behavior. Intriguingly, we found that the mouse TfAP-2 and Twz homologues, AP-2β and Kctd15, co-localize in areas of the brain known to regulate feeding behavior and reward, and a proximity ligation assay (PLA) demonstrated that AP-2β and Kctd15 interact directly in a mouse hypothalamus-derived cell line. Finally, we show that in this mouse hypothalamic cell line AP-2β and Kctd15 directly interact with Ube2i, a mouse sumoylation enzyme, and that AP-2β may itself be sumoylated. Our study reveals how two obesity-linked homologues regulate metabolic homeostasis by modulating consummatory behavior