2 Ubiquinone function in mitochondrial respiratory chain Ubiquinone = Coenzyme Q = CoQ10COCH2CHCH3Ubiquinone function in mitochondrial respiratory chainUQC ICIICIIICIVcSuccinateO2CVNADHADPATPH2Oinner membraneintermembrane spacematrixThe mitochondrial RC is composed of five complexes. Electron transfer occurs in complexes I to IV and CV is the ATP synthase allowing ATP synthesis in the mitochondria.Coenzyme Q (CoQ, ubiquinone) is a lipophilic component located in the inner mitochondrial membrane. It consists of a benzoquinone ring and a polyprenyl chain. In human ubiquinone contains 10 prenyl and is therefore called CoQ10. Ubiquinone has a pivotal role in oxidative phosphorylation. Indeed, CoQ transfers electrons from complex I and complex II to complex III.CoQ also plays a critical function in antioxidant defenses.Mitochondrial diseases are caused by a deficiency in the RC and represent a relatively common cause of metabolic diseases and the frequency of these disorders is around 1/8000 live birth. Among the RC deficiencies, we and others have observed ubiquinone deficiency.
3 Quinone biosynthesis pathway mevalonatedimethylallyl-PPisopentenyl-PPtyrosinegeranyl-PP4-OH-phenylpyruvatetranstransfarnesyl-PPprephenategeranylgeranyl-PPchorismateCHOHO4-OH-benzoatePOO-CH2CHCH3nCpolyprenyl-PPThe Q biosynthesis pathway is a relatively complicated pathway. Ubiquinone is composed of a benzoquinone ring, synthesized from tyrosine, and a polyprenyl side-chain, generated from acetyl-coA through the mevalonate pathway. The length of the prenyl chain is species specific, in human the chain consists of 10 isoprene, in rat and mouse of nine isoprene, in S. cerevisiae 6 isoprene. The transprenyltransferase, the enzyme that elongates this chain, is responsible for determining the number of isoprene units.The resulting decaprenyl –OH-benzoate then undergoes several modifications such as hydroxylations, O-methylations, methylations and decarboxylation to give ubiquinone.polyprenyl-OH-benzoateCOCH2CHCH3Ubiquinone
4 Quinone biosynthesis pathway mevalonateS. cerevisiaedimethylallyl-PPisopentenyl-PPtyrosinegeranyl-PP4-OH-phenylpyruvatetranstransfarnesyl-PPprephenategeranylgeranyl-PPchorismateCHOHOCOQ14-OH-benzoatePOO-CH2CHCH36Chexaprenyl-PPQuinone biosynthesis pathway has been extensively studied in yeast and bacteria. In the yeast S. cerevisiae ten genes (COQ1 to COQ10) encoding enzymes and proteins involved in Q synthesis have been identified. COQ1 encodes the transprenyltransferase allowing elongation of the prenyl chain. COQ2 encodes the 4-HB transprenyltransferase catalyzing the attachment of the polyisoprenoid side chain to the 4-HB ring. COQ3 to COQ7 allow modifications of the aromatic ring to give the final ubiquinone. Finally COQ8 COQ9 and COQ10 have a yet unknown function but result in a Q-deficient phenotype when mutated.COQ2hexaprenyl-OH-benzoateCOQ4COQ8COQ9COQ10COQ3COQ6COQ5COQ7UbiquinoneCoQ6
5 Quinone biosynthesis pathway mevalonateH. sapiensdimethylallyl-PPisopentenyl-PPtyrosinegeranyl-PP4-OH-phenylpyruvatetranstransfarnesyl-PPprephenateFPPgeranylgeranyl-PPchorismateCHOHOPDSS1+PDSS24-OH-benzoatePOO-CH2CHCH310Cdecaprenyl-PPMammalian homologues of the yeast and bacteria COQ genes have been identified via sequence homology. All the known yeast genes have human homologues. Some of these homologues were demonstrated to functionally complement the corresponding yeast null mutants indicating that the yeast Q biosynthesis pathway is conserved in humans.All the proteins encoded by these genes are predicted to be mitochondrially located.Finally, these genes represent obvious candidate genes for Q deficiency.COQ2decaprenyl-OH-benzoateCOQ4COQ8COQ9COQ10ADCK1ADCK2ADCK4ADCK5COQ3COQ6COQ5COQ7UbiquinoneCoQ10
6 Genes for ubiquinone deficiencies DeafnessMental retardationObesityD308Emevalonateisopentenyl-PPdimethylallyl-PPtyrosine4-OH-benzoatedecaprenyl-PPdecaprenyl-OH-benzoateCoQ10geranyl-PPtranstransfarnesyl-PPgeranylgeranyl-PPDeafnessNephrotic syndromeMental retardationMyopathyAtaxiaCataract…S382LPDSS1PDSS2Neurological distressLiver failureNephrotic syndromeN401fsX415COQ2By systematic study of the various genes involved in Q biosynthesis we also identified COQ2 and COQ8 mutations and demonstrated by functional studies in yeast that the mutations are indeed disease-causing. I will not describe all the work but I only would like to emphasize the heterogeneous clinical presentations of Q deficiencies.PDSS1 mutations result in a relatively mild disease with deafness, mild mental retardation and obesity.PDSS2 mutations were found in patients with a more severe disease as the patients presented a multiorgan involvement including encephalomyopathy and nephropathy.COQ2 mutations result in a fatal infantile multi-organ disease as the patients died in the first week of life.Finally, we and others found COQ8 mutations in patients less severely affected.PDSS2 mutations were also reported in one additional family as well as COQ2 mutations by the group of DiMauro. But the small number of families unfortunately hampers genotype-phenotype correlation. Nevertheless, the tissue specific expression of Q deficiencies and the various clinical presentations is intriguing as all the genes involved in ubiquinone biosynthesis are ubiquitously expressed.This highly variable clinical presentation is a hallmark of all RC deficiencies but until now, no explanation has been found.E551KR213WG272VG272Dc.[ insG]AtaxiaSeizuresMild mental retardationCOQ8/CABC1
7 Treatment of ubiquinone deficiency Patient with PDSS2 mutationBefore treatment Wheel-chair bound Neck muscle weakness Could not grasp Reduced visual contact Drooling, cataract Needed a nap in afternoonAfter 14 mths of treatment: Can stand and walk unaided Head control Can take, hold and give back Fixes and smiles Diseappeared Does not want to go to bed at nightWhatever the underlying mutation, patients with quinone deficiency should be given ubiquinone therapy. For some patients reported with quinone deficiency such treatment resulted in a better general condition as ability to walk, decreased lactatemia, or ataxia improvement.We have followed the effect of oral quinone treatment on a patient with PDSS2 mutation.Before treatment, at 12 years of age, the patient was wheel-chair bound and after 14 months of therapy the boy could stand, walk unaided, and ride his bicycle for more than 3 km. His bodyweight, muscle bulk, head control, and precise movements also improved. Drooling and cataract completely resolved. The patient is now 20 but he is still mentally retarded and has myopathy.
8 Treatment of ubiquinone deficiency Patient with COQ8 mutationP1: cerebellar ataxiaseizurestrunk hypotoniaoral CoQ10 treatment (5-10 mg/kg/day) no clinical benefitsCH2CH2OHCOCH3OCH3Idebenoneoral idebenone treatment (10 mg/kg/day) failed to improve his condition and worsened the course of the diseaseThis patient with COQ8 mutation was also given CoQ10 therapy. The treatment started when he was 3 and already had a neurological disease and a slight intellectual regression . This treatment had no clinical benefits.CLIC One year later he was put on short chain quinone idebenone, which not only failed to improve his condition but even worsened the course of the disease and was therefore discontinued after 7 months. The same effect has been also reported for another patient with COQ8 mutations.He was then put on ubiquinone again with no clear benefits.Coenzyme Q10UbiquinoneCOCH2CHCH3x 10CH3OH
9 Treatment of ubiquinone deficiency Patients: clinical improvement in some casesPatients mitochondria: rapid activation of CII+III activity by exogenous quinoneYeast coq mutants: rescue of growth by quinone supplementationThe respiratory chain deficiency is rapidly corrected by exogenous quinoneInsufficient uptake of CoQ10 across the blood-brain barrier in patientsNevertheless, the clinical improvement observed in some patients, the rapid activation of RC activities in muscle mitochondria by exogenous quinone and rescue of yeast mutant by quinone supplementation suggest that the RC deficiency is rapidly corrected by exogenous Q. Therefore, the absence of spectacular improvement following oral quinone supplementation in some patients suggests an insufficient uptake of CoQ10 across the blood-brain barrier.
10 Friedreich ataxia Progressive cerebellar ataxia Lack of deep tendon reflexesHypertrophic cardiomyopathyDiabetes mellitus (10%)Carbohydrate intolerance (20%)Autosomal recessiveFrequence: 1/50,000Gene localisation: 9q13-q21(Chamberlain et al, Nature 1988)The gene encodes a 210 AA protein, frataxin(Campuzano et al, Science 1996)GAA repeat expansion in the first intron
11 Iron-sulfur synthesis in yeast CIA machineryCdf1, Nbp5, Nar1Yeast Yfh1 = Human frataxinSextramitochondrialFe-S proteincytosolmitochondriaSSsq1Jac1Mge1SSmitochondrialFe-S proteinGrx5AlaYfh1Yah1Arh1NADHIsu1/2Nfs1CysIsa1/2Nfu1Mrs3/4from R. LillFe2+
16 85% The effect of idebenone oral supplementation (6 months) on the left-ventricular mass index in 52 FRDA patientsIncreased>20%15%Stable ordecreased<20%39%46%85%Decreased>-20%
17 What about the neurological condition in FRDA patients treated by idebenone ?Patients, families and/or clinicians often report:- Decreased fatigability- Improvement of delicate movements (handwriting,drawing,control of the wheelchair commands)- Better voice (use of the phone)- Decreased swallowing difficultiesYet ataxia and deep tendon reflexes did not change significantlyafter 1 year treatment
18 TRIALING IDEBENONE IN FRDA 3 patients / 6 m treat. / decreased cardiac hypertrophy / no improvement of ataxia(1998 Hôpital Necker, Paris, France; The Lancet)8 patients / 1 y treat. / scores of ARS scale improved in all patients(2001 Hospital Sant Joan de Déu, Barcelona, Spain; Arthur et al. Euromit 5)9 patients (5 treated) / 6 weeks treat. / neither improvement of cardiac hypertrophynor of neurological condition(2001 St Josef Hospital, Bochum, Germany; Schöls et al. Neurosc. Lett)11 patients / 1 y treat. / decreased heart hypertrophy in all patients / no improvement of ataxia(2001 Hôpital Sainte-Justine, Montréal, Canada; Emond et al, WebSite)29 patients (15 treated) / 6 m treat. / reduced heart hypertrophy / no improvement ofthe ARS scale(2001 Milano, Italy; for ataxia (Mariotti et al. J Neurol.)38 patients / 6 m treat. / decreased cardiac hypertrophy (50% of the patients) / noimprovement of ataxia(2002 Hôpital Necker, Paris, France; Heart; Free Rad. Res.)50 patients / 1 y treat. / decreased cardiac hypertrophy / no improvement of ataxia(2002 The French official trial; Hôpital de la Salpetrière et Hôpital Necker, Paris, France)
19 Quinone for RC deficiency treatment Restoring electron flow replacement therapy in ubiquinone biosynthesis defectsubiquinone (CoQ10)Increasing antioxidant defenses evidence of mitochondrial oxidative stress ubiquinone biosynthesis defectsidebenone