primary mitochondrial disorders due to impaired oxidative phosphorylation ( oxphos ) are a well - established cause of severe disability and precocious death in both children and adults ( koopman et  al . , 2012 ) .
no effective therapies are currently available for these conditions , but encouraging results have recently been obtained by stimulating mitochondrial biogenesis acting on either the ppar system ( wenz et  al . , 2008 ) or the amp - kinase ( ampk)/pgc1 axis ( viscomi et  al . , 2011 ) .
importantly , these approaches can in principle be extended to several mitochondrial diseases with different genetic causes , as they do not point to the correction of a specific defect but are based on a more general strategy aimed at increasing the overall residual activity of the respiratory chain .
additional targets able to activate the mitochondriogenic program and boost mitochondrial function are sirtuins 17 ( houtkooper et  al . , 2012 ) , the mammalian orthologs of the yeast silent information regulator ( sir ) 2 gene ( imai et  al . , 2000 ;
sirtuins have different subcellular localization , sirt1 and sirt6 being mainly found in the nucleus , sirt2 in the cytosol , sirt3sirt5 in mitochondria , and sirt7 in the nucleolus .
sirtuins are important regulators of several proteins , including key metabolic players , acting as either deacetylases or adp - ribosylases ( houtkooper et  al . , 2012 ) .
the most - investigated member of the family is sirtuin1 ( sirt1 ) , a nad - dependent type iii nuclear deacetylase that utilizes nad as a cosubstrate to remove acetyl groups from lysine residues of a target protein .
known targets of sirt1 are the tumor suppressor p53 , the myocyte - specific enhancer factor 2 ( mef2 ) , the forkhead box o ( foxo ) , and pgc1 , all of which regulate transcriptional programs related to increased mitochondrial function ( andreux et  al . , 2013 ) .
sirt1 activity is directly regulated by nad availability , by substrate - dependent activation , raising the hypothesis that nad acts as a metabolic sensor .
for instance , both nad levels and sirt1 activity increase in mammalian tissues in response to energy / nutrient stresses such as exercise ( cant and auwerx , 2009 , 2010 ) and fasting ( cant and auwerx , 2010 ; chen et  al .
recent studies have shown that sirt1 activation can prevent diet - induced obesity in mice .
this effect was achieved by increasing the content of nad in cells and tissues essentially through ( 1 ) dietary supplementation of suitable nad precursors , such as nicotinamide riboside , nr ( cant et  al . , 2012 ) , or ( 2 ) inhibition of nad - consuming enzymes , such as the poly(adp - ribose ) polymerase 1 , parp1 ( bai et  al . , 2011 ) .
here , we have tested the therapeutic efficacy of these strategies on a genetic mitochondrial disease model , the sco2 knockout / knockin ( sco2 ) mouse ( yang et  al . , 2010 ) .
sco2 encodes a metallochaperone involved in the formation of the copper redox centers into nascent complex iv ( cytochrome c oxidase , cox ) ( leary et  al . , 2009 ) .
mutations in this gene lead to infantile fatal encephalocardiomyopathy ( papadopoulou et  al . , 1999 ) .
most of the patients  carry at least one allele encoding the common mutation p.e140k , corresponding to the p.e129k mutation in the knockin murine sco2 allele .
the second knockout allele in this animal model is functionally null ( yang et  al . , 2010 ) .
while homozygous knockout individuals are embryonic lethal , the sco2 mice show a predominantly myopathic phenotype characterized by  exercise intolerance and associated with ubiquitous cox deficiency .
to test the effects of persistent increase of the nad pool on  the sco2 mouse , we first crossed it with a constitutive  parp1 mouse ( de murcia et  al . ,
1997 ) , which shows increased levels of nad in skeletal muscle ( bai et  al . , 2011 ) .
the sco2-parp1 double mutants showed reduced fasting blood glucose levels , body weight and epididymal white adipose tissue ( wat ) compared to sco2 littermates , whereas no differences were observed between wt and parp1 littermates ( see figure  s1 available online ) .
the endurance motor performance of sco2-parp1 double mutants , sco2 , parp1 , and wt littermates ( four animals per group ) was monitored weekly by a standard treadmill test for 4  weeks starting at 2  months of age . while sco2 mice showed markedly reduced motor performance compared to wt littermates throughout the observation time , the double mutant individuals performed as well as the wt and parp1 littermates ( figure  1a ) .
biochemically , significant reduction of complex iv activity and , to a lesser extent , complex iii as well ( yang et  al . , 2010 ) , was measured in skeletal muscle of sco2 mice . in the muscle homogenate of sco2-parp1 double mutants ,
these activities were comparable to that of parp1 and wt littermates , whereas complex i and ii activities were even higher ( figure  1b ; table s1 ) .
accordingly , the intensity of the histochemical staining specific to cox was increased in skeletal muscle of the double mutants , compared to the sco2 mutants ( figure  1c ) .
the activities of complex iii and iv ( figure  1d ; table s2 ) were also increased in the brains of double mutants relative to sco2 animals , but remained significantly lower than those measured in parp1 and wt littermates ; the histochemical reaction to cox was concordant with the biochemical data ( figure  1e ) . to test the effects of pharmacological intervention , we first administered nr to sco2 mice and wt littermates ( n  = 4/group ) , as a food admix ( 400  mg / kg ) ( cant et  al . , 2012 ) for four weeks .
metabolic parameters in the treated groups , including reduced blood glucose , plasma fatty acids , and epididymal wat , mirrored those of parp1 mice ( figure  s2 ) , confirming that nr , or its derivative nmn , was pharmacologically active in  vivo .
nr - treated sco2 mice significantly improved their motor performance compared to the vehicle - treated sco2 group , rapidly achieving the levels of motor endurance displayed by treated and untreated wt mice ( figure  2a ) , which showed no difference to each other .
these results suggest that nr treatment increases mitochondrial function in the sco2 mice through nad activation of sirt1 ( cant et  al . , 2012 ) .
accordingly , the nad / nadh ratio was significantly increased ( figure  2b ) , and the ratio between acetylated and total foxo1 , a direct target of sirt1 , was clearly reduced ( figure  2c ) in skeletal muscle of nr - treated versus vehicle - treated sco2 and wt animals .
we did not observe differences in mtdna content ( data not shown ) and citrate synthase ( cs ) activity ( figure  2f ) , but mrna levels of several genes related to either fatty acids oxidation ( fao ) , including acox and  cd36 , or oxidative phosphorylation ( coxi , coxii , coxiv , coxva ) were significantly increased in nr - treated versus vehicle - treated sco2 but not in wt animals ( figure  2d ) .
tfam , a key factor of mtdna transcription , was also increased , and ucp3 and pdk4 , which were downregulated in sco2 mice , returned to control levels upon nr treatment .
as expected , we found no change in transcripts specific to pgc1 , which is activated posttranscriptionally by sirt1 , and to two pgc1 partners , nrf1 and nrf2 .
accordingly , western blot immunovisualization demonstrated increased levels of several nuclear- and mitochondrial - dna - encoded oxphos - related proteins in nr - treated versus vehicle - treated skeletal muscle samples ( figure  2e ) . in the same specimens , mitochondrial respiratory chain activities were significantly increased ( figure  2f ; table s3 ) in nr - treated versus vehicle - treated sco2 mice , but not in wt animals .
the histochemical staining for cox reflected the biochemical results ( figure  2 g ) . in both c.  elegans and mammalian cells
, nr - dependent sirt1 activation can induce the mitochondrial unfolded protein response ( mtupr ) , a stress - response protective mechanism which can improve mitochondrial function ( durieux et  al .
we found that the mtupr - specific transcripts clpp , hsp60 , and sod2 were significantly increased in muscle samples of nr - treated sco2 mice , whereas sod3 , which is unrelated to mtupr , was unchanged ( figure  2h ) .
no effect of the nr treatment was observed on the mitochondrial respiratory chain activities in the brain of our animals ( table s4 ) .
next , we administered a pan - parp inhibitor ( mrlb-45696 , ic50 for parp  < 1  nm ; pirinen et  al . , 2014 , in this issue of cell metabolism ) at 50  mg /
kg as a food admix for 4  weeks . in mrlb-45696-treated sco2 mice , we observed metabolic effects similar to those of nr treatment ( figure  s3 ) .
weekly treadmill tests showed progressive increase , up to normal , of the motor endurance in mrlb-45696-treated sco2 , whereas no change was seen in wt mice ( figure  3a ) .
the nad / nadh ratio was significantly increased in treated versus untreated wt , but not in the sco2 , animals ( figure  3b ) , whereas the acetylated / total foxo1 ratio was reduced in both treated groups ( figure  3c ) , indicating activation of sirt1 by mrlb-45696 .
similar to nr , mrlb-45696 increased the mrna expression levels of oxphos- and fao - related genes in both sco2 and wt mice , whereas mtdna content ( data not shown ) and cs activity ( figure  3f ) remained unchanged . in treated sco2 but not in wt animals , western blot analysis showed increased content of mitochondrial respiratory chain subunits ( figures 3d and 3e ) , which was paralleled by significantly increased mitochondrial respiratory chain activities ( figure  3f ; table s3 ) .
histochemistry for cox showed increased staining in treated versus vehicle - treated sco2 mice ( figure  3 g ) .
again , we found that expression of the mtupr genes hsp60 , clpp , and sod2 was significantly increased , unlike the mtupr - unrelated gene sod3 ( figures 3h and 3i ) .
in contrast to the nr treatment , the mrlb-45696 treatment determined significant increase of cox transcripts ( figure  4a ) ,  and respiratory chain activities in the brain ( figure  4b ; table  s4 ) . the intensity of cox staining was increased as well ( figure  4c ) .
similar results were obtained in both skeletal muscle and brain by using pj34 , a commercially available pan - parp inhibitor ( figures s3 and s4 ; table s4 ) .
the nad pool is set by the balance between de novo and  salvage biosynthetic pathways and utilization by nad - consuming enzymes .
nad is synthesized de novo from tryptophan , but the main source of nad is from salvage pathways ( houtkooper et  al . , 2010 ) .
these require the uptake of other nad precursors from the diet , including nr . upon its entry in the cell , nr is phosphorylated by nr kinases into nicotinamide mononucleoside ( nmn ) , which is then converted to nad by nmn adenylyltransferase ( bieganowski and brenner , 2004 ) .
nad biosynthesis and cellular levels are also controlled by a circadian clock related to the feeding / fasting cycle .
for example , in the mitochondrial compartment nad levels regulate sirtuin 3 , a deacetylase targeting respiratory chain subunits ( peek et  al . , 2013 ) , while in the nucleus nad is a substrate of both parp1 and sirt .
parp1 , the highest consumer of nad in mammalian tissues , is activated upon binding to damaged or abnormal dna ( durkacz et  al . , 1980 ) and catalyzes the formation of poly(adp - ribose ) polymers ( par ) using nad as a substrate , onto different acceptor proteins , including parp1 itself ( adamietz , 1987 ) .
ablation of the parp1 gene , supplementation of nr or administration of parp inhibitors ( parpi ) can expand the nad pool and activate sirtuins , particularly sirt1 , a master regulator of mitochondrial homeostasis .
these effects can protect mice from high - fat ( hf)-induced metabolic disease ( cant et  al . ,
we have shown here that these treatments can correct the biochemical and clinical phenotype of the sco2 mouse , a model of genetically determined mitochondrial disease . increased transcription of genes related to both oxphos and mtupr
was associated with activation of oxidative metabolism , increase of mitochondrial respiratory chain activities , and normalization of the endurance motor deficit , displayed by naive sco2 animals .
notably , these effects were hardly seen in wt littermates , suggesting that mitochondrial dysfunction sensitizes muscle and possibly other tissues to activators of mitochondriogenic programs .
increased mitochondrial function can be achieved in wt animals only by much longer - term treatments ( > 6  months ) ( pirinen et  al . , 2014 ) .
in contrast with previous results , we found no change in mtdna copy number and cs activity in nr- or parpi - treated versus untreated animals , possibly because of the shorter timeframe of our experimental protocol ( 4  weeks ) compared to that of  other studies ( 12  weeks ) .
this observation suggests time - dependent activation of different mitochondriogenic programs , with induction of oxphos- and fao - related genes occurring much  earlier than stimulation of mitochondrial proliferation and increase in mtdna content .
likewise , prolonged nr supplementation up to 6  months induced mitochondrial biogenesis in the brain and improvement of cognitive dysfunction of an alzheimer disease mouse model ( gong et  al . , 2013 ) .
while we observed hardly any effect of nr in our 4  week trial , two pan - parp inhibitors did correct the respiratory chain defect in the brains of our sco2 mice .
this observation is particularly relevant , as the brain is an exquisite target of mitochondrial dysfunction , and progressive encephalopathy is the most frequent clinical presentation of mitochondrial disease in infancy and childhood .
our work supports the idea that the increase of nad levels in critical tissues is an effective therapeutic option for mitochondrial disease .
nr is a natural vitamin with no known adverse effects , which could be administered as a dietary supplement , particularly in case of isolated mitochondrial myopathy .
our results are concordant with very recent works reporting beneficial effects of nad precursors in mouse models characterized by reduced nad / nadh ratio , such as aging ( gomes et  al . , 2013 ) or complex i deficiency ( karamanlidis et  al . , 2013 )
initially shown to boost oxidative metabolism in diet - induced models of obesity , parp1 ablation or inhibition has recently been reported to remarkably rescue pharmacological models of liver cirrhosis , partly by correcting the associated mitochondrial impairment ( mukhopadhyay et  al . , 2013 ) .
several parp inhibitors are currently under clinical trial as anticancer molecules , and seem to be associated with relatively mild side effects ( bundred et  al . , 2013 ; tutt et  al . ,
however , more work is needed to evaluate their use in chronic conditions such as primary mitochondrial disorders in view of their potential genotoxic effects .
mouse tissues were homogenized in 15 volumes of 10  mm potassium phosphate buffer ( ph 7.5 ) .
mitochondrial - enriched fractions were collected after centrifugation at 800  g for 10  min in the  presence of protease inhibitors , and frozen and thawed three times in liquid nitrogen .
aliquots , 70  g each , were run through a 12% sds - page and electroblotted onto a  nitrocellulose membrane , which was then matched with different antibodies .
carlo besta  neurological institute , in accordance with guidelines of the italian ministry of health .
mice were maintained in a temperature- and humidity - controlled animal - care facility , with a 12  hr light / dark cycle and free access to water and food .
a standard treadmill apparatus ( columbus instruments , columbus , oh ) was used to measure motor exercise endurance , as described in viscomi et  al .
series of 8  m thick sections were stained for cox and sdh , as described ( sciacco and bonilla , 1996 ) .
muscle quadriceps samples stored in liquid nitrogen were homogenized in 10  mm phosphate buffer ( ph 7.4 ) , and the spectrophotometric activity of ci , cii , ciii , and civ , as well as cs , was measured as described ( bugiani et  al . , 2004 ) .
note that in all panels the activity of cii has been multiplied by 10 for  visualization clarity .
nad was extracted using acidic and alkaline extraction methods , respectively ( yang and sauve , 2006 ) .
tissue nad was analyzed with mass spectrometry as previously described ( yang and sauve , 2006 ) .
mtdna content and transcripts analysis was carried out by sybr green real - time pcr , as described ( viscomi et  al . , 2011 ) .
 summarymitochondrial disorders 
 are highly heterogeneous conditions characterized by defects of the mitochondrial respiratory chain . pharmacological activation of mitochondrial biogenesis has been proposed as an effective means to correct the biochemical defects and ameliorate the clinical phenotype in these severely disabling , often fatal , disorders . 
 pathways related to mitochondrial biogenesis are targets of sirtuin1 , a nad+-dependent protein deacetylase . as nad+ boosts the activity of sirtuin1 and other sirtuins , intracellular levels of nad+ play a key role in the homeostatic control of mitochondrial function by the metabolic status of the cell . 
 we show here that supplementation with nicotinamide riboside , a natural nad+ precursor , or reduction of nad+ consumption by inhibiting the poly(adp - ribose ) polymerases , leads to marked improvement of the respiratory chain defect and exercise intolerance of the sco2 knockout / knockin mouse , a mitochondrial disease model characterized by impaired cytochrome c oxidase biogenesis . 
 this strategy is potentially translatable into therapy of mitochondrial disorders in humans . 
Introduction
Results
Discussion
Experimental Procedures