the nervous system relies on conserved endocytic and synaptic vesicle ( sv ) recycling mechanisms to sustain high activity .
defective endocytosis and sv recycling have long been implicated in neurodegeneration and neurodegenerative diseases , yet the molecular mechanisms remain unclear ( esposito et  al . , 2012 , heutink and verhage , 2012 , saheki and de camilli , 2012 , schreij et  al . , 2016 ) .
endophilin - a ( henceforth endophilin ) endocytic adaptors are key players in membrane dynamics at the neuronal synapse ( ringstad et  al . , 1999 ,
, 2002 , milosevic et  al . , 2011 , saheki and de camilli , 2012 ) .
endophilin-1 and 3 are brain - specific , whereas endophilin-2 is ubiquitous ( ringstad et  al . , 1997 , ringstad et  al . , 1999 ) .
all three contain a highly conserved bar domain and have a central role in both clathrin - mediated endocytosis ( farsad et  al . , 2001 ,
, 2001 , verstreken et  al . , 2002 , milosevic et  al . ,
2011 ) and clathrin - independent endocytosis ( boucrot et  al . , 2015 ) . in clathrin - mediated endocytosis , endophilin recruits synaptojanin-1 to the necks of coated vesicles prior to scission by dynamin ( milosevic et  al . , 2011 ) .
furthermore , availability of endophilin is rate limiting for vesicle uncoating and sv recycling , and neurons without endophilin show reduced neurotransmission ( milosevic et  al . , 2011 ) .
total absence of endophilin is perinatally lethal , whereas mice lacking endophilin-1 and 2 ( 1,2 double knockout [ dko ] ) show ataxia , neurodegeneration , and early lethality ( milosevic et  al . , 2011 ) .
it can not be easily explained by defective neurotransmission , because even mice with paralysis and abolished neurotransmission do not develop neurodegeneration ( heutink and verhage , 2012 ) .
thus , it is unlikely that neurodegeneration in endophilin 1,2 dko mice is solely the result of defective sv recycling .
mice without endophilin also show increased protein ubiquitination ( cao et  al . , 2014 ) , and neurodegeneration
is frequently associated with impaired proteolytic systems , most notably the ubiquitin - proteasome system ( ups ) and autophagy ( nakamura and lipton , 2009 , nixon , 2013 , menzies et  al . , 2015 ) .
endophilin has been linked to the ups through direct interaction with the parkinson s disease ( pd)-related e3-ubiquitin ligase parkin ( trempe et  al . , 2009 ) and as a key component of the cbl - cin85-endophilin complex known to downregulate receptors ( soubeyran et  al .
additional links between endophilin and neurodegeneration have recently emerged . the most commonly disrupted gene in  familial pd ( cookson , 2010 ) , lrrk2 , encodes a protein that phosphorylates endophilin ( matta et  al . , 2012 ) .
lrrk2 appears in lewy bodies and plays a role in apoptosis and autophagy ( iaccarino et  al .
in addition , a  number of case studies described synaptojanin-1 and auxilin mutations in early - onset parkinsonism ( edvardson et  al . , 2012 , quadri et  al . , 2013 , krebs et  al . , 2013 ) .
auxilin is recruited to the clathrin - coated vesicles directly after the action of synaptojanin-1 , which is brought to the neck of the clathrin - coated pit by endophilin ( milosevic et  al . , 2011 ) .
interestingly , endophilin is upregulated in the brains of pd patients ( shi et  al . , 2009 ) .
in  addition , endophilin-1 and 3 have been shown to interact with ataxin-2 , mutations in which lead to spinocerebellar ataxia type  2 ( ralser et  al . , 2005 , nonis et  al . , 2008 ) . despite direct parkin - endophilin interaction and a striking upregulation of parkin in mice lacking all three endophilin genes ( endophilin triple knockout [ tko ] ; cao et  al . , 2014 ) , elevated parkin
we subjected endophilin mutants to rna sequencing and found upregulation of another e3-ubiquitin ligase , f - box - only protein 32 ( fbxo32 , also called atrogin-1/mafbx ) .
fbxo32 has previously been studied in muscles , where its induction occurs prior to the onset of rapid atrophy ( sandri et  al . , 2004 ) .
while studying how the fbxo32-endophilin - a interaction balances protein degradation and neuronal health in the brain , we discovered a direct role of endophilin - a in autophagosome formation .
mice lacking all three endophilin genes die perinatally , whereas endophilin 1,2 dko animals show ataxia , neurodegenerative changes , and lethality around 3  weeks ( movie s1 ; milosevic et  al . , 2011 ) . whereas single knockout ( ko ) and heterozygous ( 1ht-2ht ; 1ht-2ht-3ht ) mice appear phenotypically normal , endophilin 1ko-2ht , 1ko-2wt-3ko , and particularly 1ko-2ht-3ko
mice exhibit shorter lifespans ( 19 , 21 , and 18  months , respectively ; 1ht-2ht mice lived over 27  months ; figure  s1a ) . observing 1ko-2ht and
1ko-2ht-3ko mice revealed progressive impairments in motor coordination and increasingly obvious ataxia as these animals age . to systematically measure the progression of ataxia , we used a composite phenotype scoring system that incorporates hindlimb clasping , ledge walking , kyphosis , and gait ( guyenet et  al . , 2010 ) .
when applied to four endophilin lines ( 1ko-2wt , 1ko-2ht , 1ko-2wt-3ko , and 1ko-2ht-3ko ) and wild - type ( wt ) controls , it revealed phenotypic differences between endophilin mutants and within the same animals over the course of aging ( figure  1a ) ; i.e. , the onset and progression of ataxia correlate with age and severity of endophilin deficiency .
next , we systematically evaluated locomotion by accelerating rotarod ( arr ) performance test and gait analysis . whereas the performance of 1ko-2wt mice on the arr was comparable to wt , fall latency in 1ko-2ht-3ko mice was already significantly lower at a very young age ( figure  1b ) .
intermediate phenotypes were observed in 1ko-2ht and 1ko-2wt-3ko mice , where fall latency compared to wt was decreased significantly at 3 and 15  months , respectively .
gait analysis revealed that ability to run on a motorized treadmill and walking pattern also depended on the number of missing alleles and age ( figures 1c , 1d , and s1b ; see movie s2 ) .
1ko-2ht-3ko gait was impaired at 3  months , whereas the littermate 1ko-2wt-3ko mice showed impaired gait at 15  months ( figure  1d ) .
overall , onset and progression of movement impairments correspond to age and the extent of endophilin deficiency . to evaluate whether neurodegenerative changes could cause these motor impairments , we inspected neuronal cell death and gliosis in our model . whereas overall neuronal cell morphology was unaltered in these mice ( figure  s1d ) , tunel assay revealed slightly increased cell death in endophilin 1ko-2wt and more prominently in 1,2 dko motor cortex and hippocampus at postnatal day ( p)18 ( figure  1e ) . increased cell death was also found in 18-month 1ko-2ht-3ko motor cortex and hippocampus ( figure  s1c ) .
glial fibrillary acidic protein immunostaining revealed prominent upregulation of gliosis in 1,2 dko motor cortex ( figure  1f ) . because endophilin tko mice die a few hours after birth , we probed for early markers of apoptosis in endophilin tko brains and found an increase in caspase 3 activity ( figure  1 g ) . in summary , newborn endophilin tkos , juvenile 1,2 dko , and aged 1ko-2ht-3ko mice display signs of mild to moderate neurodegeneration .
we were interested in the molecular mechanisms underlying neurodegeneration in our endophilin - deficient models , so we employed rna sequencing ( experimental strategy in figure  s2a ) .
we first sequenced hippocampal rna from tko mice that show altered sv recycling and controls ( n  = 89 mice / condition ) .
the data are of high technical quality ( 27 m aligned reads ) and samples clustered well according to genotype ( table s1 ; figures s2b and s2c ) .
182 differentially expressed genes ( degs ) between endophilin tko and littermate controls and 964 degs between tko and wt were detected ( adjusted p value  < 0.01 ; log2 fold change > 0.4 ) ( tables s2 and s3 , respectively ) .
given the pronounced movement defects of endophilin mutants , we first looked for movement - disorder - related genes or pathways .
interestingly , tko versus wt degs showed significant enrichment of genes involved in ataxia and pd ( figures 2a and 2b ; table s4 ) . because endophilin 1,2 dko mice present first signs of neurodegeneration by the end of the 2 postnatal week , hippocampi from dko , littermate 1ko-2wt , and wt mice were extracted during the 3 week and subjected to rna sequencing and transcriptome analysis .
the data are again of high technical quality ( 28 m reads ; table s1 ; samples clustered well according to genotype ; figures s2d and s2e ) .
1,479 degs between 1,2 dko and littermate controls , and 1,749 degs between dko and wt were detected ( adjusted p value  < 0.01 ; log2 fold change  > 0.4 ; tables s5 and s6 , respectively ) . both 1,2 dko versus littermate and dko versus wt lists were significantly enriched for genes involved in ataxia and pd ( table s4 ; figures 2c and 2d ) .
ataxia - related genes include atm interactor ( atmin ) , e2f1 , and rad51 ; pd - related genes include park2 ( encoding parkin ) , lrrk2 , the tyrosine hydroxylase gene ( th ) , and parl . these findings further support endophilin s association with neurodegeneration , ataxia , and pd ( arranz et  al . , 2015 , edvardson et  al . , 2012 ,
, 2013 , matta et  al . , 2012 , milosevic et  al . , 2011 , quadri et  al . , 2013 , ralser et  al .
, 2005 , shi et  al . , 2009 , trempe et  al . , 2009
we next analyzed tko versus both littermate and wt controls ( genes significantly changed in the same direction in tko against both littermates and wts ; table s7 ; figure  s2f ) by ingenuity pathway analysis ( ipa ) software to identify altered pathways and networks .
the resulting most - represented pathways were related to compromised synaptic transmission , protein homeostasis , phagosome maturation , mitochondrial function , dopamine metabolism , and apoptosis ( figure  2e ) . altered protein homeostasis has been strongly linked to neurodegeneration ( e.g. , deger et  al .
, 2015 ) and might be relevant to the ataxic and neurodegenerative phenotypes observed in endophilin - deficient mice .
the differential expression of several genes was validated by  quantitative real - time pcr in endophilin tko hippocampi versus wt , and a strong correlation was observed ( r  = 0.83 ; figure  2f ) .
we also examined regulators of the transcriptional profile in endophilin tko hippocampi and found that , among those statistically enriched , fbxo32 presents the most - robust change in gene expression ( 1.9-fold increase ; table s8 ) .
this was interesting because fbxo32 is an e3-ubiquitin ligase linked to autophagy in skeletal and cardiac muscle ( sandri et  al .
its expression is known to be regulated by the transcription factors foxo1 and foxo3a ( sandri et  al . , 2004 ,
webb and brunet , 2014 ) : whereas foxo1 was unchanged , foxo3a was significantly upregulated in tko hippocampi ( table s8 ) .
given the observed changes in the foxo3a - fbxo32 network and that fbxo32 is known to promote cell death and is required for muscle atrophy ( sandri et  al .
, 2004 , wu et  al . , 2011 , xie et  al . , 2009 )
, we decided to follow up on this network at the rna and  protein level .
as in the rna - sequencing data , foxo3a and fbxo32 were upregulated in tko hippocampi when measured by qpcr ( figure  3a ) whereas foxo1 was unchanged ( figures s2 g and s2h ) .
a striking increase in fbxo32 protein levels in tko whole - brain extracts relative to littermate controls and to wt mice was detected ( figure  3b ; figure  s2i displays elevated fbxo32 in the hippocampus ) .
levels of foxo3a[ser253 ] phosphorylation ( which represses foxo3a binding to dna ) were also  decreased ( figure  3b ) , suggesting an increase in the  foxo3a transcriptional activity , and the distribution of foxo3a[ser253]-p is altered in tko hippocampus in comparison to controls ( figure  3c ) . to determine whether foxo3a activation is directly responsible for the upregulation of fbxo32
, foxo3a was silenced in tko primary hippocampal neurons by small interfering rna ( sirna ) knockdown , using two sirna constructs .
near - complete ablation of foxo3a affects neuronal survival , so we used a milder condition ( 45% reduction ) to probe for fbxo32 levels ( figure  3d ) .
the elevated fbxo32 levels in tko neurons were lowered upon foxo3a knockdown , implying that foxo3a is a sufficient regulator of fbxo32 in this system ( figure  3d ) .
fbxo32 was also strongly upregulated in 1,2 dko brain samples , both at p0 ( whole brain ) and 3  weeks ( cortex ; figure  3e ) , as well as in the cortex of 1ko-2ht-3ko mice at 18  months ( figure  3f ) .
thus , the foxo3a - fbxo32 network originally described in  muscle atrophy is operative in the brain of endophilin mutant mice . given the reported role of fbxo32 in the regulation of protein homeostasis via the ups , we investigated whether the ubiquitin system is also affected in endophilin mutants .
the pd - related e3-ubiquitin ligase parkin monoubiquitinates endophilin , and tko brains show increased levels of parkin and ubiquitinated proteins ( trempe et  al . , 2009 , cao et  al . , 2014
however , 1,2 dko mice in which the parkin gene park2 was also knocked out displayed a similar neurological phenotype to 1,2 dko mice ( cao et  al . , 2013 ) , suggesting that other ubiquitin ligases might be involved .
given that fbxo32 is also an e3-ubiquitin ligase , we hypothesized that its robust upregulation in endophilin - deficient brain might contribute to the loss of protein homeostasis in endophilin knockouts .
first , we tested whether fbxo32 can ( mono)ubiquitinate endophilin-1 and 2 , but this was not the case ( figures s3a and s3b ) .
we observed that 1,2 dko brains also display increased levels of ubiquitinated proteins ( figure  4a ) , similar to tko brains
( cao et  al . , 2013 ) . to distinguish whether this was due to proteasome - related or localization-/signaling - related ubiquitination , we measured k48-(proteasome - related ) and k63-(signaling - related)-linkage - specific polyubiquitination ( herhaus and dikic , 2015 ) . whereas k48-linked ubiquitination was prominently increased in 1,2 dko cortical extracts ( figure  4b )
, there was no significant change in the levels of k63-linked ubiquitination ( figure  4c ) , suggesting that the accumulation of ubiquitinated proteins is associated with defective processing by the proteasome .
given the apparent inability of the proteasome to cope with the  level of ubiquitinated proteins in endophilin knockouts , we probed the proteasomal system using a ubiquitinated substrate that is targeted to the proteasome and degraded ( ubiquitin - g76v - gfp ; dantuma et  al . , 2000 ) , as well as by measuring the enzyme activity of the proteasome itself .
we observed an accumulation of gfp - fluorescence originating from the substrate in tko primary hippocampal neurons as well as fibroblasts ( mouse embryonic fibroblasts [ mefs ] ) in comparison to respective controls ( figures 4d and 4e ) , denoting proteasome saturation in the endophilin ko cells .
interestingly , we noticed that the increase in ubiquitinated proteins elicited by the expression of ubiquitin - g76v - gfp results in significantly more apoptosis in the tko mefs than in controls ( figure  4 g ) .
altogether , these results suggest a causal link between proteasome saturation in endophilin mutants , higher levels of ubiquitinated proteins , and increased cell death susceptibility . to determine whether the proteasome saturation was due to  decreased activity of the proteasome , we measured the proteasome enzyme activity .
while there was a trend toward a decrease in proteasomal activity , we observed no significant  difference in the proteasome activity per se in the brain extracts ( figure  4f ) .
thus , the proteasome system in tko brains seems functional but unable to keep up with the high demand . in light of the upregulation of fbxo32 in the absence of endophilin
first , we tested whether fbxo32 interacts with endophilin family members by immunoprecipitation and pull - downs .
an interaction between fbxo32 and all three endophilins in  vivo was revealed by specific enrichment of fbxo32 in anti - gfp immunoprecipitates from hela expressing fbxo32-mcherry and endophilin 1 , 2 , or 3-egfp ( figures 5a5c ) .
similar results were obtained by glutathione s - transferase ( gst ) pull - downs from mouse brain lysate using the gst - fbxo32 as bait ( figure  s3e ) .
we demonstrated that fbxo32 is able to pull - down purified endophilin-1 protein ( figure  5d ) , suggesting a direct interaction .
furthermore , the interaction is mediated through endophilin s sh3 domain , as revealed by another set of pull - downs from mouse brain lysate using the gst - sh3 domains of endophilin-1 and 3 as baits ( an enrichment of fbxo32 with stronger binding to endophilin-1 and 3 sh3s was observed ; figure  s3f ) .
we examined the intracellular localization of fbxo32 and its  overlap with endophilins in cultured clonal cells ( hela and  mefs were used to control for cell line variability ) and primary hippocampal neurons .
it is noteworthy that expressing fbxo32 ( either fbxo32-egfp or fbxo32-mcherry ) induced cell death : 50% of fbxo32-transfected
hela and 40% of fbxo32-transfected mefs survived 30  hr post - transfection ; less than 15% of fbxo32-transfected primary neurons were detected on day in  vitro ( div ) 14 ( detailed below ) .
fbxo32 distribution showed substantial cell - to - cell variability : although mainly cytosolic , fbxo32 was enriched at dynamic organelle - like structures of various sizes and often accumulated in the nucleus ( movie s3 ) .
native fbxo32 distribution was similar to that of expressed fbxo32 : immunostaining against fbxo32 revealed nuclear as well as cytosolic protein distribution in hela cells ( figure  s4a ) .
surprisingly , both in hela cells and mefs , fbxo32-labeled tubular structures were observed ( examples in figures 5f , 5i , and s4c ; see also movie s5 ) . given that fbxo32 interacts with endophilins , and the propensity of endophilins to tubulate membranes , we examined the colocalization of fbxo32 with endophilin 1 , 2 , and 3 .
the distribution of endophilins is mainly cytosolic : sparse puncta were attributed to endophilin s role in endocytosis ( milosevic et  al .
, 2011 , perera et  al . , 2006 , boucrot et  al . , 2015 ,
when endophilin-1 and fbxo32 were coexpressed at similar levels , fbxo32 protein distribution changed : fbxo32 became predominantly cytoplasmic and excluded from the nucleus ( figures s4b and s6e ; also see figures 7b and 7d ) .
interestingly , we also observed that cell death due to fbxo32 overexpression was abolished upon endophilin coexpression , which we detail further below . fbxo32 and endophilin-1
were also found to colocalize sparsely at dynamic organelle - like structures ( figures 5l and s4b ) .
these were found throughout the cytoplasm and not  only in the vicinity of the plasma membrane ( as would be expected for endophilin-1 when being recruited to endocytic structures ) . when endophilin-2 and fbxo32 were coexpressed , they colocalized transiently at both organelle - like structures and tubuli ( figures 5e , 5l , and s4c ; movie s5 ) .
we further observed that endophilin-2 colocalized with fbxo32 on dynamic tubulated structures , although endophilin-2 was not needed for the recruitment of fbxo32 to tubuli ( figure  5f at 24 s ; movie s5 ) .
similarly to endophilins , fbxo32 also directly binds and tubulates membranes in  vitro ( b. kroppen , m. meinecke , and i.m .
lastly , fbxo32 and endophilin-3 colocalized partially on organelle - like structures ( figures 5l and s4d ) .
we examined the overlap between fbxo32 and endophilin-1 or endophilin-2 in primary hippocampal cultures . fbxo32-egfp and endophilin-1-mrfp or endophilin-2-mrfp signal overlapped partially both in the cell bodies and in neuronal processes ( figures 5 g and 5h ; again fbxo32 colocalized to a greater extent with endophilin-2 than endophilin-1 ) . in sum , the data show that fbxo32 interacts directly with endophilin - a proteins ( possibly through their sh3 domain ) , both proteins are involved in membrane tubulation , and both transiently colocalized on dynamic organelle - like structures of various sizes .
in addition to being an e3-ubiquitin ligase , fbxo32 is known to regulate autophagy in muscle , raising the question of whether the fbxo32-labeled organelle - like structures were autophagosomes .
we coexpressed fbxo32-egfp together with lc3-mrfp and observed a prominent colocalization of the two probes in starved cells ( figures 5i and 5 m ; movie s4 ) .
a similar result was observed when fbxo32 was immunostained ( figure  s4a ) , irrespective of type of cell ( hela cells were also used ) or fluorescent labeling ( figure  s4e ) .
next , when endophilin-2 was coexpressed with the autophagosomal marker lc3 , sparse but significant colocalization was observed in starved hela cells , suggesting that endophilin-2 , like fbxo32 albeit to a lesser extent , transiently associates with autophagosomes ( figures 5j and 5 m ) .
similar observations were made for endophilin-1 and endophilin-3 , which were also occasionally seen on lc3-labeled autophagosomes ( figures 5 m , s4f , and s4 g ; please note that the values represent colocalization above coincidental colocalization , which is prominent in this system given that endophilins are cytosolic proteins ) .
for example , a kymograph from endophilin-3- and lc3-expressing hela cell shows that endophilin-3 colocalizes with an lc3-labeled autophagosome transiently ( figure  5n ; arrows indicate colocalization ) .
importantly , fbxo32 , lc3 , and endophilin-2 could be simultaneously detected at the same dynamic organelle ( movie s6 ) .
notably , endophilin-2 showed much stronger overlap with atg5 ( an early autophagosomal marker ) than with lc3 ( figure  5k ; 43% colocalization ; see movie s7 ) , implying a role for endophilin - a in autophagosome formation .
given that both fbxo32 and endophilins - a were occasionally found on lc3-positive autophagosomes , we examined whether autophagy was altered in brains of tko and 1,2 dko mice .
notably , both lc3b - ii ( lower band is the active form ) , a classical marker for autophagosomes , and atg5 , a marker for early autophagosomes , were prominently decreased in tko brains ( figure  6a ) and in aged endophilin 1ko-2ht-3ko mice ( figures 6c and 6d ) .
interestingly , the atg5 protein level in endophilin 1ko-2ht-3ko mice decreases progressively between 3 and 18  months of age ( figure  6d ) . a reduction in lc3b - ii and atg5
was also observed in 1,2 dko cortex ( figure  6b ) , suggesting that endophilin-3 can not effectively compensate for the loss of endophilin-1 and 2 in autophagy .
we prepared primary neuronal cultures from endophilin tko and wt hippocampi and labeled autophagosomes either by mildly expressing lc3-egfp ( figures 6e and s5c ) or by immunostaining for lc3 ( figure  s5e ) .
we detected an overall decrease in the number of lc3-labeled autophagosomes when  autophagy was simultaneously induced by starvation and mtor inhibitor torin-1 ( figures 6e , 6f , s5c , and s5d ) .
interestingly , the effect was more obvious at the synapses , as apparent from colocalization of lc3 and synaptobrevin-2 , a presynaptic marker ( figure  6e ; insets in figure  s5c ) .
altogether , the biochemical and imaging data suggest that autophagy depends on endophilin in  vivo and in  vitro and imply a role for endophilin in the formation and/or maturation of autophagosomes .
notably , endophilin - b has a known role in autophagosome formation , and its cdk5-mediated phosphorylation is required for induced autophagy in models of pd ( wong et  al . , 2011 ) .
endophilin - a proteins are closely related to endophilin - b : they share an identical domain structure with a high homology , and both protein subfamilies are enriched at the synapses and able to tubulate membranes ( farsad et  al . , 2001 ,
thus , we tested the levels of endophilin - b , cdk5 , and several autophagy initiators .
no change in the protein level of endophilin - b in endophilin - a tko brains was detected ( figure  6 g ) , whereas the levels of cdk5 were decreased ( figure  6a ) .
however , in 1,2 dko brains , the cdk5 level was unchanged ( figure  6b ) .
protein levels of uvrag , beclin , atg3 , and hdac6 were not altered , either in endophilin tko brains or endophilin 1,2 dko cortex ( figures s5a and s5b ) .
these results imply that endophilin - b can not compensate for autophagosome formation in the absence of endophilin - a and that autophagy is not altered due to repression of signaling pathways , because the key autophagy initiation regulators are not affected by absence of endophilin - a . the key autophagy factors atg9a and atg16l have been shown to undergo clathrin - mediated endocytosis ( ravikumar et  al .
, 2010a , puri et  al . , 2014 ) . in the light of endophilins
well - established  role in endocytosis , it is possible that the observed phenotype is an indirect effect of defective endocytosis in endophilin mutants .
however , we found that both atg9a and atg16l were not significantly changed in our mutants ( figures 6h and s5f ) , indicating that availability of these two factors does not influence the findings we describe here . to further characterize the role of fbxo32 in autophagy , we employed the drosophila melanogaster neuromuscular junction ( nmj ) to see whether fbxo32 is relevant for autophagy in neuronal cells in  vivo . when fbxo32 is silenced in drosophila in  vivo , there was a very obvious decrease in the number of autophagosomes detected at the nmj ( figures s6a  s6c ; quantification in figure  s6d ) .
thus , fbxo32 seems necessary for autophagosome formation at the fruit fly nmj . given that , in the endophilin mutant brains there is more fbxo32 and yet still less autophagosome formation
, the interaction between endophilin and fbxo32 may play a key role in this process . to determine whether upregulation of fbxo32 is an important step in the brain pathology of endophilin mutants
, we explored the consequences of fbxo32 upregulation in clonal ( hela and mef ) and primary ( neurons ) cell lines .
it has been reported that fbxo32 elevation induces apoptosis in cardiomyocytes and cancer cells ( wu et  al . , 2011 , xie et  al . , 2009 ) .
we observed that expression of fbxo32 causes substantial cell death in hela and mefs , as determined by the number of remaining egfp - positive cells and  by the activity of caspases 3 and 7 ( figures 7a7c , 7e , and s6e ) .
the detrimental effects of fbxo32 were independent of its fluorescent tag and cell - transfection method ( electroporation / lipofectamine / fugene ) .
strikingly , when endophilin-1 was coexpressed with fbxo32 in  mefs , fbxo32-induced cell death was rescued , as demonstrated by the number of egfp - positive cells ( normalized [ norm . ] to total dapi count ) 30  hr after transfection and healthy cell appearance ( figure  7b ; also in hela cells : figure  s6e ) , the quantification of cell survival ( figure  7c ) , and the activity of caspases 3 and 7 ( figure  7e ) .
importantly , the levels of fbxo32 expression were elevated when fbxo32 and endophilin-1 were coexpressed , implying that the rescue of fbxo32-induced apoptosis is not due to a reduction in fbxo32 levels ( figures 7a and 7b ) .
however , coexpression with endophilin-1 seemed to relocate fbxo32-egfp out of the nucleus ( figures 7d and s6e )  in agreement with the data presented in figure  5 .
thus , fbxo32-induced apoptosis is associated with aberrant intracellular fbxo32 localization , and both the localization and the apoptosis susceptibility can be rescued by simultaneous upregulation of endophilin-1 .
these data show that fbxo32 and endophilin-1 interact not only at the protein but also at the genetic level . in neurons ,
when either wt primary hippocampal or cortical neurons were transfected with fbxo32-egfp , less than 15% of all transfected neurons were detected at div14 ( figures 7f7h ) .
the surviving fbxo32-expressing neurons displayed a lower number of processes and appeared unhealthy ( figure  s6f ) .
tko hippocampal and cortical neurons in  culture were comparable to wt and littermate endophilin 1ko-2wt-3ko cells and did not show increased cell death  under standard culturing conditions .
however , when tko cortical neurons were transfected with fbxo32 , less than 2% of transfected neurons were detected at div14 ( in comparison to egfp - expressing tko neurons ) , with the surviving cells presenting a reduction in  the  number of processes and vacuoles in cell bodies .
lastly ,  coexpression of  endophilin-1 or endophilin-2 was able to rescue  fbxo32-induced apoptosis in  cortical neurons : when endophilin-1-mrfp or endophilin-2-mrfp were coexpressed with fbxo32-egfp , either in wt and endophilin tko cortical neurons , the majority of neurons thrived ( over 80% and 67% of all fbxo32-transfected neurons , respectively ; figures 7f7h ) .
imbalance in fbxo32/endophilin - a levels is likely a key mechanistic step in the brain pathology of endophilin mutants , and fbxo32-endophilin interplay seems important for maintaining neuronal health and overall protein homeostasis in the brain .
to date , the known roles of endophilin - a have been limited to endocytosis and sv recycling . here , we show that endophilin also affects the ups and autophagy in the brain .
the robust induction of the foxo3a / fbxo32 network ( known to mediate muscle atrophy ) in the brain of mice with endophilin deficiency promotes apoptosis and likely mediates brain pathology .
similarly to muscle , the foxo / fbxo32 network seems relevant for regulating protein degradation in the brain and for maintaining neuronal health .
if fbxo32 is expressed in cell lines and primary neurons , it causes apoptosis that can be rescued by simultaneous coexpression of endophilin .
we also report that partial endophilin absence in mice results in neurodegeneration and in perturbations of a significant number of ataxia- and pd - related genes .
these observations corroborate a number of recent findings that indirectly link endophilin to neurodegeneration and pd ( arranz et  al . , 2015 , edvardson et  al . , 2012 , krebs et  al . , 2013 , matta et  al . , 2012 , milosevic et  al . , 2011 ,
endophilin models may also be of relevance for neurodegeneration research because they may provide insights into signaling pathways affected prior to overt manifestation of neurodegeneration .
fbxo32 was thus far studied mainly in skeletal muscle , where this e3-ubiquitin ligase was found to mediate rapid atrophy as a result of foxo3a transcriptional activation .
interestingly , foxo3a activation is associated with cellular stress responses and neurodegeneration ( webb and brunet , 2014 ) .
foxo3a stimulates autophagy in neurons ( xu et  al . , 2011 ) , and
fbxo32 is necessary for autophagy ; activation of the foxo - fbxo32 axis should thus lead to increased autophagy .
nevertheless , this is not the case when endophilin is lacking : the number of autophagosomes is sharply decreased .
also , endophilin colocalizes transiently with mature and even more impressively with early autophagosomes , suggesting that endophilin - a has a previously unreported role in the formation and/or maturation of autophagosomes .
this is supported by the recent identification of endophilin - a as one of the regulators of autophagosome formation ( strohecker et  al . , 2015 ) .
interestingly , mice without endophilin also display the same pattern of perinatal lethality observed in mice lacking proteins necessary for autophagosome formation ( kuma et  al . , 2004 ) .
given endophilin s involvement in endocytic processes , the decrease in the number of autophagosomes might also result from compromised endocytosis .
specifically , it has been shown that blockage of endocytosis upstream of clathrin coat assembly results in decreased autophagosome formation ( ravikumar et  al .
. however , endophilin deficiency results in the accumulation of clathrin - coated vesicles , and the levels of clathrin are not altered ( milosevic et  al . , 2011 ) .
thus , clathrin is available to interact with atg16l1 , and rna and protein levels of atg16l1 are not altered in this model .
in addition , the rna and protein levels of atg9 , a key autophagic protein known to traffic via the plasma membrane and endosomal compartments ( mari et  al . , 2010 , puri et  al . , 2014 ) are not altered in endophilin mutant brains . altogether
, whereas we can not entirely exclude endophilin s role in endocytosis also affecting autophagy , we consider other mechanism(s ) besides endocytic defects to be underlying the decrease in autophagosomes in endophilin mutants . decreased autophagosome availability and , consequently
, lower autophagic flux are known to affect protein homeostasis ( korolchuk et  al . , 2009 ) .
we observed an accumulation of k48-linked ubiquitinated proteins in the cytoplasm , suggesting saturation of the ups ( due to overloading rather than deficiencies in the  proteasome ) .
there is possibly a double hit on this pathway : decreased autophagy and increased availability of ubiquitin ligases ( fbxo32 and parkin ) .
both fbxo32 and parkin are involved in selective autophagy of ubiquitinated proteins ( zaglia et  al . ,
2014 ) , and both function under the regulation of foxos ( webb and brunet , 2014 ) .
upregulation of fbxo32 and parkin may thus be a compensatory response to defective autophagy , an attempt to shunt more of the burden to the ups , or possibly both .
interestingly , the lower abundance of autophagosomes in the absence of endophilin - a can not be ( fully ) compensated for by a related protein with the same domain organization , endophilin - b , an established mediator of autophagosome formation ( takahashi et  al . , 2011 ) .
endophilin - a primarily operates in the endocytic processes at the plasma membrane , whereas endophilin - b has  prominent roles in apoptosis , mitochondrial membrane dynamics , and autophagosome formation ( kjaerulff et  al . , 2011 ) .
furthermore , cdk5-mediated phosphorylation of endophilin - b is required for inducing autophagy in models of pd ( wong et  al . , 2011 ) .
more research is needed to understand endophilin - a and endophilin - b links to autophagy , neurodegeneration , and pd .
we propose a model in which endophilin s absence , likely due to its membrane - curvature - inducing / stabilizing properties , leads to reduced availability of membranes for autophagosome formation and decreased autophagic activity .
interestingly , fbxo32 and endophilin interact at both the gene and protein level and both are required for the autophagy process .
like endophilin , fbxo32 can tubulate membranes and is recruited to membrane tubules in  vivo . thus
, endophilin and fbxo32 membrane - tubulating activity may contribute to the formation / maturation of autophagosomes .
this model is in concordance with the reported role of endophilin - a as necessary and sufficient for macroautophagy at presynaptic terminals of fruit flies ( soukup et  al .
in summary , our study addresses the origin of neurodegeneration in a key mammalian endocytic model and reveals that the foxo / fbxo32/endophilin network may play a role in maintaining neuronal health by balancing autophagy and the ups in the brain .
the results are consistent with the following plausible scenario : reduced endophilin availability results in impaired capacity to form autophagosomes , resulting in decreased autophagy , accumulation of ubiquitinated proteins in the cytoplasm , proteasome saturation , and further accumulation of unwanted proteins over time that eventually affect neuronal function and health , leading to neurodegeneration .
accordingly , we show that imbalanced autophagy also develops in aged endophilin heterozygote mice that display age - dependent progressive ataxia .
our data reveal a role in autophagy for a key endocytic adaptor previously thought to be strictly dedicated to endocytosis and invite further research addressing the physiological and pathological implications of these findings .
supplemental experimental procedures detail gene - targeting and cloning strategies and list the antibodies and plasmids used .
animal experiments were conducted according to the european guidelines for animal welfare ( 2010/63/eu ) with approval by the lower saxony landesamt fr verbraucherschutz und lebensmittelsicherheit ( laves ) , registration number 14/1701 . for rotarod experiments ,
620 males / group were tested on a setup by tse systems ( 3375 - 5 ) . a training phase consisted of four trainings over 2  days ( intertrial interval [ iti ] was 25  hr ; 180  s at 5  rpm ) .
the testing phase consisted of four tests over 2  days ( iti 25  hr ) .
mice were placed on the rod , which then accelerated from 5 to 40  rpm in 180 s. fall latency was measured .
a simple composite phenotype scoring system for evaluating mouse models of ataxia was applied as described ( guyenet et  al . ,
eight to twelve mice were used per condition , and scoring was performed by two researchers independently .
ventral plane videography for gait analysis was performed using instrumentation ( digigait ) and software from mouse specifics , according to manufacturer s instructions . for each group ,
tunel assay was performed on cryosections of the brains of perfused mice to test for apoptotic and necrotic cells .
mice were anesthetized with chloral hydrate ( 40  mg / ml ) before cardiac perfusion with 4% paraformaldehyde ( pfa ) .
after slow dehydration with sucrose , brains were embedded in oct ( tissue tek ) and frozen in liquid n2 .
sagittal brain sections were cut with a cryostat ( 10  m thick ) . sections were post - fixed and permeabilized with 0.1%
in situ cell death detection kit ( fluorescein ) was used according to manufacturer s instructions .
slides were counterstained with dapi , embedded with mowiol 4 - 88 , and imaged with a lsm 710 confocal setup ( zeiss ) .
immunofluorescence of frozen brain sections and cultured neurons ( div 1420 ) was carried out as described ( ringstad et  al . , 2001 , milosevic et  al . , 2011
live mefs , hela cells , and neurons were imaged either using a zeiss axioobserver z1 total internal reflection fluorescence ( tirf ) microscope with an evolve charge - coupled device ( ccd ) camera ( photometrics ) and 100 plan apo zeiss objective or perkin elmer ultraview spinning - disk confocal setup that consists of nikon ti - e eclipse inverted microscope equipped with  perfect focus , 60 cfi plan apo vc nikon objective , and 14-bit electron - multiplied ccd camera ( hamamatsu c9100 ) .
autophagy induction and colocalization analysis was performed as detailed in the supplemental experimental procedures and below .
whole - brain or cortex samples for western blotting were prepared by homogenization of brain tissue ( whole brain for p0 animals ; one cortex for p14p21 animals and aged animals ) as detailed in supplemental experimental procedures . for trap - gfp immunoprecipitation
, hela cells were transfected with endophilin ( 1 , 2 , or 3)-egfp and mcherry / fbxo32-mcherry using lipofectamine 3000 ( invitrogen )
. we used chromotek trap - gfp agarose beads ( chromotek ) and followed manufacturer s protocol . alternatively , hela cells were transfected with fbxo32-egfp and coupled to chromotek trap - gfp agarose beads .
purified endophilin-1 was added , and all fractions ( input , non - bound , and bound ) were collected .
primary hippocampal and cortical cultures were prepared as previously described ( ferguson et  al . , 2007 ) .
primary neurons expressed constructs , following amaxa ( lonza)-based transfection , under the control of the chicken--actin promoter to allow for long - term expression .
a minimum of 15 images from three to five experiments were analyzed for each genotype / condition .
given that the majority of respective fluorescent signals were cytosolic , colocalization was evaluated semi - automatically using object - based overlap analysis and imagej jacop plugin ( bolte and cordelires , 2006 ) . in short , the image was first segmented into objects and background ( i.e. , bright fluorescent objects are segmented from the image ) , and then their spatial relationship ( overlap ) was measured .
the same analysis was performed with the mirror image , and random colocalization was subtracted in each case .
, circles were superimposed on bright fluorescent spots in the fbxo32 ( or lc3 or atg5 ) channel and transferred to identical image locations in the endophilin channel .
if the fluorescence intensity maximum in the endophilin channel was located in the same circle and the morphology of the signal resembled that of the fbxo32 signal , the circle was rated as positive ( colocalized ) ; if not , it was rated as negative ( not colocalized ) . to correct for accidental colocalization , circles were also transferred to a mirror image of the endophilin channel ( detailed in supplemental experimental procedures ) .
we used wt and endophilin tko mefs , immortalized with e6/e7 viral proteins , grown on supplemented dmem at 5% co2 at 37c .
cells were pelleted and resuspended with n - dodecylmaltoside 1.5% in pbs with complete protease inhibitor cocktail ( roche ) and phosstop phosphatase inhibitor cocktail ( roche ) and lysed at 4c in a rotator for 30  min . the lysate
was then centrifuged to pellet debris and placed in 96-well glass - bottomed plates for use in the assay . the fluorimetric proteasome assay ( proteasome 20s activity assay kit ) was performed according to manufacturer s instructions using a synergy h1 plate reader ( biotek ) at 37c .
caspase 3/7 apoptosis assay was performed with the apotox - glo triplex assay ( promega ) kit according to a modified version of the manufacturer s protocol for 96-well plates , detailed in supplemental experimental procedures .
figures 2e [ with n.r . ] , 3a , 3b , 3e , and 6a6c ; c.m.r . : figures 1 [ with i.m . ] , 3c , 3f , 4a4c , 6d , 6 g , and 6h ) ; supervision , p.v .
 summaryendophilin - a , a well - characterized endocytic adaptor essential for synaptic vesicle recycling , has recently been linked to neurodegeneration . 
 we report here that endophilin - a deficiency results in impaired movement , age - dependent ataxia , and neurodegeneration in mice . 
 transcriptional analysis of endophilin - a mutant mice , complemented by proteomics , highlighted ataxia- and protein - homeostasis - related genes and revealed upregulation of the e3-ubiquitin ligase fbxo32/atrogin-1 and its transcription factor foxo3a . 
 fbxo32 overexpression triggers apoptosis in cultured cells and neurons but , remarkably , coexpression of endophilin - a rescues it . fbxo32 
 interacts with all three endophilin - a proteins . similarly 
 to endophilin - a , fbxo32 tubulates membranes and localizes on clathrin - coated structures . additionally , fbxo32 and endophilin - a are necessary for autophagosome formation , and both colocalize transiently with autophagosomes . 
 our results point to a role for endophilin - a proteins in autophagy and protein degradation , processes that are impaired in their absence , potentially contributing to neurodegeneration and ataxia . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions