a common feature of aged organisms is functional alteration and gradual loss of muscle mass and force .
a deficit of muscle innervation due to motor - neuron loss is believed to have a major contribution to age - dependent muscle wasting , also called sarcopenia .
this age - acquired deficit in muscles contributes profoundly to reduced quality of life in elderly people and predisposes them to an increased risk of morbidity , disability , and mortality ( visser and schaap , 2011 ) .
the etiology of sarcopenia is multifaceted and involves several intrinsic and extrinsic factors . despite the clinical , social , and economic relevance of sarcopenia ,
the precise mechanisms for the age - related loss of muscle mass and function are not yet fully understood .
age - related changes in muscle are complex , with key features including myofiber atrophy , profound weakness that is partially independent of muscle mass loss , myofiber degeneration , accumulation of dysfunctional mitochondria , and increased oxidative stress . until recently , it was thought that age - associated atrophy and weakness were secondary to motor - neuron loss in the brain or in the spinal cord .
in fact , little neuronal death occurs in most areas of the aging nervous system , and there is no decline of lower motor neurons during aging ( chai et  al . , 2011 ; morrison and hof , 1997 ) .
conversely , it is emerging that neuromuscular junctions ( nmjs ) and their interactions with myofibers are greatly altered during aging , resulting in a loss of muscle innervation ( chai et  al .
oxidative stress and decreased release of trophic factors are independent influences on nmj integrity and contribute to denervation ( jang et  al .
however , to date , it is not known whether myofiber denervation is due to deleterious changes in muscle cells themselves , in neurons , or both .
two lifestyle adaptations , caloric restriction and exercise ( jang et  al . , 2012 ; valdez et  al . ,
2010 ) , have been consistently demonstrated to extend lifespan and , in parallel , to mitigate age - related alterations of nmj .
macroautophagy , hereafter named autophagy , is activated by both of these conditions in skeletal muscles and other tissues ( grumati et  al . , 2010 , 2011a , 2011b ; he et  al . , 2012 ; rubinsztein et  al .
autophagy is a highly conserved homeostatic mechanism used for the degradation and recycling of bulk cytoplasm , long - lived proteins , and organelles through the lysosomal machinery ( mizushima and komatsu , 2011 ) .
the autophagy machinery generates double membrane vesicles ( autophagosomes ) that engulf and sequester target cellular components ( mizushima and komatsu , 2011 ) .
therefore , autophagy can be defined as a process of cytosolic renovation . in mammals ,
deficient expression of atg1 , atg7 , atg8 , becn1 ( beclin1 ) , and sestrin1 ( which is also required for basic autophagy ) shortens the lifespan of the fruit fly d.  melanogaster and of the nematode c.  elegans ( lee et  al . , 2010b ; rubinsztein et  al . , 2011 ; simonsen et  al . ,
2008 ) . on the other hand , increased autophagy contributes to longevity , and mutation of essential atg genes prevents the gain of longevity ( melndez et  al . , 2003 ) .
a recent report shows that aging can be controlled by a single tissue ; overexpression of foxo transcription factor or its target eif4ebp1 in fly skeletal muscle abolishes the age - associated decline in autophagy and increases longevity ( demontis and perrimon , 2010 ) .
, 2007 , 2008 ; zhao et  al . , 2007 ) , and genetic variations in the human foxo3a
the role of autophagy in sarcopenia in mammals is still controversial . both impaired and excessive autophagy have been associated with aging sarcopenia ( wenz et  al . , 2009 ; wohlgemuth et  al . ,
however , loss - of - function experiments that support the involvement of autophagy in aging sarcopenia are still lacking . here
, we show that specific inhibition of autophagy in skeletal muscle leads to abnormalities in motor neuron synapses that ultimately lead to denervation whereas accumulation of dysfunctional mitochondria triggers oxidation of contractile elements , causing a decrease in force generation .
age - related muscle loss is believed to result from a general decline of protein synthesis or an enhancement of protein breakdown . to study autophagy in aging , we monitored the expression of autophagy markers , such as lc3 ( map1lc3a ) and the critical e1-like enzyme , atg7 , in mice and humans .
aged mice showed a decline of atg7 protein and , concurrently , of lc3 lipidation when compared to young animals ( figure  1a ) .
an important decrease of atg7 protein and lc3ii was found in muscle biopsies of elderly sedentary ( sarcopenic ) subjects ( figure  1b ) . because exercise activates autophagy and counteracts most age - related characteristics , including the decline of muscle mass ( zampieri et  al . , 2014 )
, we tested whether long - life regular exercise in humans was able to prevent the reduction of these autophagy markers .
atg7 protein and lc3ii were maintained in muscle biopsies of senior sportsmen that had been previously shown to have better muscle mass and strength than elderly sedentary subjects ( zampieri et  al .
to mimic the decrease of atg7 in aging , we characterized muscle - specific autophagy - deficient ( atg7 ) mice that we have generated ( masiero et  al . , 2009 ) .
the absence of autophagy caused a significant reduction of animal survival ( figure  2a ) .
morphological analyses of aged mice showed higher levels of atrophy , center - nucleated fibers , and inflammation in atg7 muscles compared to age - matched controls ( figures 2b and s1a ) .
some fibers atrophied to a level  that made them hardly detectable ( figure  2c ) .
these small  fibers  did not express the typical markers of regeneration and , therefore , are adult atrophic myofibers ( data not shown ) .
quantification of the cross - sectional area ( csa ) of atg7 revealed that , although muscle atrophy was present in both oxidative and glycolytic fibers , it was more pronounced in the latter ( figures 2d and 2e ) .
the deterioration of atg7 muscles was also confirmed by the increase of center - nucleated fibers ( figure  2h ) .
correspondingly , atg7 muscles were weaker than controls both in adult and elderly mice ( figures 2f and 2 g ) . given that the features of atrophic fibers resemble denervated fibers , and a decline in innervation and loss of motor units are known to be important endogenous causes of sarcopenia , we tested whether denervation plays a role in the phenotype of atg7 mice .
we first examined the expression pattern of the neural cell adhesion molecule ( ncam ) , a molecule that is enriched in the postsynaptic membrane of the nmj but largely absent in adult myofibers .
however , ncam is re - expressed along the entire muscle fiber after the loss of innervation .
immunohistochemistry revealed that , during aging , there is a progressive increase of ncam - positive fibers in controls .
importantly , atg7 muscles showed 5- to 7-fold more ncam - positive fibers when compared to age - matched controls both in adult and elderly mice ( figures 2i2k and s1b ) .
the expression of two other markers of denervation , musk ( musk ) and the acetylcholine receptor ( achr ) -subunit ( chrng ) were significantly more upregulated in atg7 muscles ( figure  2l ) . to confirm these findings
, we used a recently described in  vivo imaging approach where two distinct achr pools are differently color labeled ( rder et  al . , 2008 , 2010 ) to monitor nmj morphology and stability .
automated image analysis revealed that , in contrast to control muscles , nmjs of adult atg7 knockout mice showed fragmented morphology ( figures 3a and 3b ) and instability ( figures 3a and 3b ) . whereas in controls , the changes in morphology and instability progressively increased with age , this was not the case in atg7 knockout mice ( figure  3b ) .
nmjs of both adult and aged atg7-deficient animals showed significantly more fragmentation and achr turnover than 26-month - old ( aged ) control animals ( figure  3b , right panel ) .
exocytic and endocytic processes mediate delivery of newly formed achr and retrieval of old achr , respectively .
inhibition of autophagy led to accumulation of vesicles close to the endplates that contain bungarotoxin ( bgt ) ( figures 3c and 3d ) , further corroborating an impairment of achr turnover and of endosome trafficking . to determine whether alteration in endosome trafficking might also affect musk , a kinase that affects a plethora of signaling pathways that are important for nmj stability , we monitored musk localization
. immunohistochemistry revealed that most musk colocalized with achr at the plasma membrane in control fibers ( figure  3e ) .
importantly , atg7 caused a significant loss of musk on the plasma membrane and a concomitant enrichment of internalized musk ( figure  3e ) .
these findings confirm an alteration of achr and musk recycling and localization in atg7 mice .
to determine whether the changes of nmj are secondary to p62 ( p62/sqstm ) accumulation into aggregates that clear signaling factors such as nrf2 or secondary to inhibition of the conjugation system , we knocked down p62 in atg7 .
for in  vivo transfection experiments , we used bicistronic vectors that simultaneously encode small hairpin rnas ( shrnas ) and gfp . in this way , detection of  gfp fluorescence allowed us to monitor the efficiency of transfection and the changes that occurred to p62-positive aggregates .
after 14  days of transfection , atg7 tibialis anterior ( ta ) muscle was cleared by p62-positive inclusions ( figure  s2a ) . however , despite the absence of p62-positive aggregates , the  percentage of denervated ncam - positive fibers was not improved ( figure  s2b ) . to understand the role of autophagy in age - related nmj alterations , we used the inducible muscle - specific atg7 knockout mice that we have recently generated ( masiero et  al . , 2009 ) .
the atg7 gene was acutely deleted in 22-month - old mice , and mice were sacrificed 3  months later .
western blots for p62 and lc3 revealed that autophagy was successfully blocked in aged mice ( figure  4a ) .
the inhibition of autophagy in old mice led to  an exacerbation of muscle mass degeneration as revealed by the increase of centronucleated fibers and the decrease of csa ( figures 4b and 4c ) .
fiber size distribution analysis revealed that acute atg7 deletion led to an enrichment of small fibers when compared to controls ( figure  s3 ) , suggesting that denervation occurred in a discrete number of fibers .
we then monitored nmj to determine whether autophagy inhibition in old mice was sufficient to destabilize muscle - nerve interaction .
atg7 deletion led to a significant increase of denervated ncam - positive fibers ( figure  4d ) .
denervation is also supported by the finding that the musk transcript is significantly induced in atg7 ( figure  4e ) .
acute inhibition of autophagy in aged mice caused a significant loss of musk on the myofiber plasma membrane and a concomitant enrichment of internalized musk protein ( figure  4f ) . to determine the involvement of autophagy in nmj maintenance during aging , we overexpressed atg7 protein in 26-month - old atg7 mice to restore normal autophagy flux in aged mice .
atg7 expression was sufficient to increase lc3-positive vesicles in sarcopenic muscles ( figure  5a ) .
when we monitored the effect of atg7 expression on nmj of aged mice , we found a significant improvement in morphology ; nmj was significantly less fragmented than age - matched controls ( figures 5b and 5c ) . correspondingly , restoring
autophagy in aged mice led to a decrease of vesicles close to the endplates that contain bgt , further corroborating a reactivation of achr turnover and of endosome trafficking ( figure  5d ) .
this was confirmed by the fact that musk localization was improved by the expression of atg7 ( figure  5e ) . rescuing atg7 protein and
autophagy led to a reduction of denervated ncam - positive fibers that returned to the values of adult mice ( figure  5f ) . finally , improvement of autophagy flux and nmj morphology resulted in an enhancement in muscle mass ( figure  5 g ) .
one potential explanation of age - related loss of muscle mass and innervation is due to accumulation of dysfunctional mitochondria that generate an enhancement of reactive oxygen species ( ros ) production and oxidative stress . over the past years
, several researchers have reported that oxidative stress might be involved in etiology of weakness , nmj degeneration , and sarcopenia ( jang et  al . , 2010 , 2012 ) .
in fact , inhibition of autophagy induced an accumulation of mitochondria as revealed by succinate dehydrogenase ( sdh ) staining ( figure  6a ) .
electron microscopy analyses revealed the presence of giant mitochondria containing abnormal cristae ( figure  6b ) .
atg7 flexor digitorum brevis ( fdb ) myofibers showed a significant increase of mitochondria that depolarized when the f1f0-atpase was blocked by oligomycin ( grumati et  al .
similarly , fdb myofibers isolated from aged atg7 showed a significant increase in depolarized mitochondria , which was corrected by restoring atg7 expression ( figure  s4a ) .
the alteration of mitochondrial morphology and function is associated with increased oxidative stress as revealed by protein carbonylation ( figure  6d ) .
atg7 muscles showed a 2-fold increase of carbonylated proteins when compared to age - matched controls .
we performed a proteomic approach on carbonylated proteins and found that mitochondrial and sarcomeric proteins are more oxidized in atg7 than controls ( table s1 ; figure  s4b ) .
because oxidative stress is believed to affect force generation , its enhancement might contribute to age - dependent weakness .
morphological and functional analyses on isolated single - skinned fibers confirmed that atg7 myofibers are more atrophic and generate less force ( figures 6e and s4c ) .
when the absolute force was normalized for myofiber size , the resulting specific force was still significantly lower ( figure  6f ) .
thus , not only the muscles became smaller , but there was a general impairment in force transmission , which led to profound weakness .
the reduction of specific force may be caused by posttranslational modifications of contractile proteins induced by ros .
we then purified actin and myosin from atg7 muscles and confirmed that both proteins were more oxidized in atg7 mice ( figure  6 g ; table s1 ) .
acto - myosin function was investigated using an in  vitro motility assay approach ( canepari et  al . , 2012 ) .
actin sliding velocity on both myosin and heavy meromyosin fraction ( hmm ) of atg7 was slower compared to controls ( figure  6h ) , confirming that an alteration of the functional properties of contractile proteins occurs when autophagy is reduced . to test the role of oxidative stress in sarcopenia , we treated animals with trolox , a potent antioxidant , for 4  weeks .
the treatment successfully reduced the amount of total carbonylation on muscle protein extracts ( figures 7a and s4b ; table s1 ) .
protein carbonylation of trolox - treated atg7 no longer differed from age - matched controls .
the proteomic approach confirmed that trolox abolished the oxidation of mitochondrial and sarcomeric proteins ( table s1 ; figure  s4b ) .
importantly , antioxidant treatment blocked the oligomycin - dependent mitochondrial depolarization of atg7 mice ( figure  7b ) .
we then addressed whether atrophy , weakness , and nmj degeneration are affected by blunting oxidative stress .
the treatment did not rescue myofiber size ( figures 7c and s5 ) but reduced the drop of specific force in isolated atg7-deficient myofibers ( figure  7d ) .
trolox completely prevented carbonylation of purified myosin and actin ( figures 7e and s4b ) and restored a normal acto - myosin function ( figure  7f ) .
looking at nmj , we found that inhibition of ros in adult atg7 mice partially reduced nmj instability whereas it did not give protection from nmj fragmentation ( figure  7 g ) . in conclusion , these findings strongly suggest that ros production directly affects acto - myosin interaction and force generation but shows a limited involvement in the changes occurring at nmj and no effect on atrophy .
the present study provides several important insights concerning the role of autophagy in nmj preservation during aging .
the most striking conclusion is that autophagy controls two important destabilizing factors that are ( 1 ) the removal of dysfunctional mitochondria that produce ros and ( 2 ) the endosome recycling of achr that affects musk localization and muscle - nerve interaction .
the consequence of aging is the deterioration of tissue function mainly due to accumulation of damaged dna , proteins , and organelles .
autophagy is a critical system to maintain the cell clear from dysfunctional organelles and mis- or unfolded proteins that are prone to aggregate and to preserve dna stability ( sandri , 2010 ) . due to these actions and
that autophagy declines with aging , there is consensus in considering autophagy as an antiaging system .
genetic evidence in flies and worms sustain this concept , but these data are still lacking in mammals .
in fact , autophagy knockout mice die as newborns due to a general energy failure ( kuma et  al . , 2004 ) , and most of the tissue - specific knockouts ( hara et  al . , 2006 ; komatsu et  al . , 2006 ;
aging is a multisystemic disorder that affects multiple organs , leading to alterations of metabolism and tissue function .
the postmitotic tissues such as brain , heart , and skeletal muscle are the most susceptible to age - related disease , and their dysfunction contributes importantly to precocious aging
. the important role of muscle in healthy aging has been sustained by recent studies in drosophila .
activation of autophagy by overexpression of foxo transcription factor in the muscles of flies increases longevity ( demontis and perrimon , 2010 ) .
the extension of lifespan is due to reduction of food intake and insulin release from neurosecretory cells , confirming that maintenance of a normal autophagy level in skeletal muscle , but not in adipose tissue , positively affects whole - body metabolism ( demontis and perrimon , 2010 ) . however , the role of autophagy in age - related muscle loss in mammals is still unclear , being either beneficial or detrimental ( gaugler et  al . , 2011 ; wenz et  al . , 2009 ; wohlgemuth et  al . ,
our findings support the notion that autophagy is critical to prevent age - related denervation and weakness .
indeed , autophagy inhibition is sufficient to trigger a cascade of events that resemble those happening in elderly people . because mitochondria are the main producers of ros , dysfunctional mitochondria are thought to play a key role in muscle function decline . in old age , a significant portion of mitochondria
are abnormally enlarged , more rounded in shape , and display vacuolization in the matrix and shorter cristae ( peterson et  al .
these features are present in autophagy - deficient muscles of adult animals , suggesting that defects in mitochondrial turnover are involved in age - dependent mitochondrial abnormalities .
it is established that ros production is increased in aged muscle in both the subsarcolemmal and intermyofibrillar pools of mitochondria ( chabi et  al . , 2008 ) .
this increase in ros has been reported to induce oxidation of complex v , leading to decreased atp production ( peterson et  al . , 2012 ) . accordingly
, adult atg7 muscles showed a significant increase of carbonylated atp synthase ( table s1 ) .
consistent with the ros - mediated inhibition of atp synthase , the energy - stress sensor of the cell , ampk , is activated in atg7-null muscles ( not shown ; masiero et  al . , 2009 ) .
this finding is consistent with a recent report that showed improvement of age - related deficit , including mitochondrial function , by expressing a mitochondrial - targeted human catalase ( lee et  al . , 2010a ) .
. in muscle , oxidation of contractile proteins has been described , but the functional meaning of this condition is unclear .
we showed by proteomic analyses that contractile proteins are carbonylated and that trolox treatment reverts both protein oxidation and the myosin capacity of moving actin filaments , ultimately improving muscle force .
it is well established that changes in muscle mass and strength tend to be dissociated in elderly persons , the decline of muscle strength being three times faster than the decrease of muscle mass ( peterson et  al . , 2012 ) .
this suggests that alteration in the quality of contractile proteins plays a critical role during age - related decrease of muscle force .
accordingly , atg7 myofibers show a significant decrease of force compared to age - matched controls , and this decline is corrected by trolox . however , whereas the antioxidant improves acto - myosin function , it does not protect from muscle atrophy , although we can not exclude that extending trolox treatment for longer periods may successfully inhibit the signaling pathways controlling muscle atrophy .
previous studies reported that mitochondrial ros production may contribute to nmj instability ( dobrowolny et  al . , 2008 ;
overexpression of uncoupling protein ( ucp1 ) , specifically in skeletal muscle , is sufficient to dismantle nmj and to induce distal motor neuron degeneration ( dupuis et  al . , 2009 ) .
in fact , whereas trolox treatment slightly reduced the turnover of old and new achr , it did not prevent nmj fragmentation . in conclusion ,
our data strongly support the concept that autophagy in a postmitotic tissue is required for the correct interplay between muscle and nerve and for the quality control of mitochondria .
our findings also sustain the concept that the beneficial effects of caloric restriction and exercise on aging are a consequence of autophagy reactivation .
animals were handled by specialized personnel under the control of inspectors of the veterinary service of the local sanitary service ( asl 16padova ) , the local officers of the ministry of health .
all procedures are specified in the projects approved by the italian ministero della salute , ufficio vi ( authorization numbers c65 ) .
subjects were male volunteers who received detailed information about the study and gave informed consent .
approval from the ethics committees of the city of vienna and the comenius university in bratislava for medical ethics was obtained at the outset of the study .
muscle - specific atg7-null ( atg7 ) and tamoxifen - inducible muscle - specific atg7 mice have been previously described ( masiero et  al . , 2009 ) .
tamoxifen - induced cre loxp recombination was activated by oral administration of tamoxifen - containing chow ( tam400/cre er harlan ) . in  vivo transfection and
sequences and plasmids are described in the supplemental experimental procedures . for in  vitro validation of shrna constructs , c2c12 cells were maintained in dulbecco s modified eagle s medium ( dmem)/10% fetal bovine serum and transfected with shrna constructs using lipofectamine 2000 ( life technologies ) .
muscle force was measured in living animals as previously described ( blaauw et  al . , 2008 ) .
the method used for single - muscle - fiber dissection , the solutions , and the experimental setup have been previously described ( bottinelli et  al . ,
single - fiber analysis was performed as previously described ( bottinelli et  al . , 1996 ) .
absolute ( po ) and specific forces ( po / csa ) of the fibers were determined . at the end of the mechanical experiment ,
fibers were put in 20  l of laemmli buffer and stored at 20c for subsequent electrophoretic analysis of myosin heavy chain ( mhc ) isoform content .
myosin isoform 2b was extracted from bulk gastrocnemius muscles of mice according to a procedure previously described in detail ( canepari et  al . , 2000 ) and used to prepare the hmm .
hmm was obtained by a proteolytic digestion with -chimotrypsin of myosin according to a modification of the method of margossian and lowey ( 1982 ) previously described in detail ( canepari et  al . , 2000 ) .
g - actin was extracted as described by pardee and spudich ( 1982 ) from acetone powder prepared from the residues of mice muscles after myosin extraction .
after polymerization , f - actin was labeled by incubation for several hours with rhodamine - phalloidine ( molecular probes r415 ) as described by kron et  al .
myosin ( or hmm ) was diluted to 0.1  mg / ml in a high ( or low ) ionic strength buffer and infused in a flow cell treated with nitrocellulose and prepared according to anson et  al .
the in  vitro motility assay ( ivma ) analysis was performed according to canepari et  al .
the composition of the experimental buffer was 3-(n - morpholino)propanesulfonic acid 25  mm ( ph  = 7.4 at 25c ) , kcl 25  mm , mgcl2 4  mm , egta 1  mm , dithiothreitol ( dtt ) 1  mm , glucose oxidase 200  g / ml , catalase 36  g / ml , glucose 5  mg / ml , and atp 2  mm .
the velocities of at least 50 filaments were measured and their distribution characterized according to parametric statistics .
complementary dna generated with life technologies superscript iii reverse transcriptase was analyzed by quantitative real - time pcr using power sybr green applied biosystem .
cryosections of frozen gastrocnemius muscles were lysed in a buffer containing 50  mm tris ( ph 7.5 ) , 150  mm nacl , 10  mm mgcl2 , 0.5  mm dtt , 1  mm edta , 10% glycerol , 2% sds , 1% triton x-100 , roche complete protease inhibitor cocktail , and sigma protease inhibitor cocktail .
the samples were immunoblotted as previously described ( sandri et  al . , 2004 ) and visualized with supersignal west pico chemiluminescent substrate ( pierce ) .
carbonylation of muscle proteins was detected by using the oxyblot protein oxidation detection kit from millipore ( masiero et  al . ,
mice were intraperitoneally injected with trolox ( 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid ) 30  mg / kg daily for 4  weeks . for a detailed description of the detection of oxidized proteins , see the supplemental experimental procedures . spot detection and quantification
was carried out using the image master 2d platinum 7 software ( ge healthcare ) . for a detailed description of the procedure ,
see the supplemental experimental procedures . for a detailed description of the procedure , see the supplemental experimental procedures .
immunofluorescence staining was performed on cryostatic sections as previously described ( masiero et  al . , 2009 ) and then monitored with a fluorescence microscope or confocal microscopy .
fiber cross - sectional area was performed on ta or gastrocnemius ( gcn ) as described ( mammucari et  al . , 2007 ) and measured using imagej software . for electron microscopy
, we used conventional fixation - embedding procedures based on glutaraldehyde - osmium fixation and epon embedding .
c2c12 myogenic cell lines were cultured in dmem ( gibco - life technologies ) supplemented with 10% fetal bovine serum .
myoblasts were transfected using lipofectamine 2000 ( life technologies ) according to the manufacturer s instructions .
mitochondrial membrane potential was measured by epifluorescence microscopy based on the accumulation of tetramethylrhodamine , methyl ester ( tmrm ) fluorescence as previously described ( romanello et  al . , 2010 ) .
the mhc isoform composition of gastrocnemius muscles of mice was assessed by 8% sds polyacrylammide gel electrophoresis as described by pellegrino et  al .
( 2003 ) . in  vivo microscopy of mice was performed under anesthesia using zoletil and xylazine on a leica sp2 confocal microscope equipped with a 63 1.2 numerical aperture water immersion objective , essentially as described previously ( rder et  al .
automated analysis of achr turnover and nmj fragmentation used algorithms described earlier ( rder et  al .
the sample size was calculated using size power analysis methods for a priori determination , based on the sd and effect size previously obtained using the experimental methods employed in the study . for animal studies , we estimated sample size from the expected number of knockout mice and littermate controls , which was based on mendelian ratios .
considering a likely drop - off effect of 10% , we set the sample size of each group at five mice . to reduce the sd , we minimized physiological variation by using homogenous animals with the same sex and age .
the exclusion criteria for animals were pre - established . in case of death , cannibalism , or sickness , the animal was excluded from analysis .
tissue samples were excluded in cases such as cryo - artifacts , histological artifacts , or failed rna extraction .
we included animals from different breeding cages by random allocation to the different experimental groups .
animal experiments were not blinded ; however , when applicable , the experimenters were blinded to the nature of samples by using number codes until final data analysis was performed .
statistical tests were used as described in the figure legends and were applied upon verification of the test assumptions ( e.g. , normality ) .
 summarythe cellular basis of age - related tissue deterioration remains largely obscure . 
 the ability to activate compensatory mechanisms in response to environmental stress is an important factor for survival and maintenance of cellular functions . 
 autophagy is activated both under short and prolonged stress and is required to clear the cell of dysfunctional organelles and altered proteins . 
 we report that specific autophagy inhibition in muscle has a major impact on neuromuscular synaptic function and , consequently , on muscle strength , ultimately affecting the lifespan of animals . 
 inhibition of autophagy also exacerbates aging phenotypes in muscle , such as mitochondrial dysfunction , oxidative stress , and profound weakness . 
 mitochondrial dysfunction and oxidative stress directly affect acto - myosin interaction and force generation but show a limited effect on stability of neuromuscular synapses . 
 these results demonstrate that age - related deterioration of synaptic structure and function is exacerbated by defective autophagy . 
Introduction
Results
Discussion
Experimental Procedures