synaptic transmission is the main energy - consuming process in the brain and requires large amounts of atp . therefore , when oxygen becomes limiting , there is a risk that the atp pool becomes exhausted . we hypothesized that neurons possess adaptive mechanisms to prevent such an energy crisis during hypoxia and speculated that hypoxia might remodel dendritic spines to reduce the energy - consuming process of synaptic transmission . the oxygen sensors prolyl hydroxylase domain - containing proteins ( phd1 - 3 ) use oxygen to hydroxylate prolines in target proteins , such as hypoxia - inducible transcription factor  ( hif ) ( epstein et  al . , 2001 ) . hydroxylated hif is ubiquitinated by the e3 ubiquitin ligase von hippel - lindau ( vhl ) , triggering proteasomal degradation ( ivan et  al . , 2001 , jaakkola et  al . , 2001 phds are inactive , and stabilized hif upregulates the transcription of target genes ( epstein et  al . , 2001 ) . phds also have hif- and hydroxylation - independent functions and targets ( wong et  al . , 2013 ) . they are implicated in synaptic plasticity , learning , and memory ( hotulainen and hoogenraad , 2010 ) . spines sprout as filopodia that search for synaptic contact and develop into mature spines containing the postsynaptic density receiving synaptic input ( ethell and pasquale , 2005 , hotulainen and hoogenraad , 2010 ) . among actin cross - linkers , filamin a ( flna ) promotes the formation of orthogonal networks or parallel actin bundles , depending on the filamin / f - actin ratio  ( van der flier and sonnenberg , 2001 ) . 2014 ) , the axonal growth cone ( zheng et  al . , 2011 ) , and neuronal migration ( sarkisian et  al . , 2006 ) , but it is unknown whether flna regulates spine morphology . notably , under- and overexpression of flna impair neuronal migration via distinct mechanisms ( sarkisian et  al . , 2006 , zhang et  al . , 2012 , zhang et  al . , 2013 ) , indicating that neurons require precise regulation of flna levels . phd2-haplodeficient endothelial cells have impaired migration and actin cytoskeleton reorganization ( mazzone et  al . , 2009 ) , whereas phd2-deficient hela cells show altered cell migration via an hif - independent mechanism ( vogel et  al . , 2010 ) . these findings raised the question whether oxygen , via phd2 , might control actin - dependent spine formation . we thus investigated the role of phd2 in the morphogenesis and maintenance of dendritic spines and synapses in hippocampal neurons . in mouse hippocampal neurons ( mhns ) , dendritic spines emerge during 811  days in  vitro ( div ) and progressively mature to shorter mushroom - shaped spines at 1215 div , with concomitant induction of spontaneous synchronized network - wide spiking activity ( figures s1a  s1e ) . to evaluate whether physiological levels of hypoxia ( which can be as low as 0.5% o2 in the brain ( ereciska and silver , 2001 ) ) affect dendritic spine formation , 13-div mhns were incubated overnight ( o / n ) in normoxia ( 21% o2 ) or hypoxia ( 1% o2 ) . to visualize dendritic protrusions , we transfected 10-div mhns with yellow ( yfp ) or tandem dimer tomato ( tdt ) fluorescent proteins . neurons exposed to hypoxia showed reduced protrusion density , and most spines were long filopodium - like protrusions without a head ( figures 1a1e ) . similar effects were seen in established mature spines when treatment started at 20 div ( figures s1f  s1j ) . time - lapse imaging of 14-div yfp - labeled mhns for 1  hr showed that , in normoxia , 45% of the spines were stable in length ( variations  0.2  m ) , whereas the rest slightly increased or reduced their length ( figures 1f1h ) . in hypoxia ( 0.5% o2 ) , most spines became longer or regressed , and only 33% kept their original length ( figures 1f1h ) . similar effects were observed upon o / n treatment of 13-div mhns with the phd inhibitor dimethyl - oxalylglycine ( dmog ) , which upregulates hypoxia - responsive genes and the hif - dependent luciferase reporter ( figures s1o and s1p and s2a  s2i ) without causing apoptosis or oxidative stress ( figures s1k  s1n ) . neurons did not suffer irreversible damage because washout of dmog restored spine density and maturation ( figures s2j  s2m ) . thus , inactivation of phds ( by hypoxia or pharmacologic inhibition ) impaired spine maturation , caused spine regression , and reduced spine density without altering viability . to evaluate whether hypoxia and dmog altered the electrophysiological properties of neurons , we performed patch - clamp recordings . 13- or 20-div mhns were incubated o / n in normoxia or hypoxia or treated with dmog . neither hypoxia nor dmog altered their passive electrical membrane properties ( figures s3a  s3c ) or intrinsic excitability ( figures s3d  s3f ) , as confirmed by evoking antidromic action potentials ( aps ) upon repeated extracellular electrical stimulation ( figure  s3 these results show that hypoxia or dmog did not change the ability of neurons to initiate and propagate aps upon stimulation . they did also not alter the time course of individual aps ( figures s3h  s3k ) . however , hypoxia and dmog reduced the frequency of spontaneous ap firing ( figures 2a and 2b ; figure s3l ) , suggesting that mhns had fewer functional synaptic connections and received less excitatory synaptic input . we also explored whether the morphological changes of dendritic protrusions were reflected by functional alterations in synaptic transmission . hypoxia and dmog reduced excitatory synaptic transmission at 14 and 21 div , as evidenced by the suppressed spontaneous synchronized network - wide spiking activity ( bursts ) , whereas inhibitory synapses were unaffected because not only burst duration but also burst frequency were reduced ( figures 2c and 2d ; figures s3m s3o ) . neurons recovered from hypoxia or dmog and restored synchronized spiking activity 24  hr after switching back to control conditions ( figures 2c and 2d ; figures s3m s3o ) , consistent with the morphological recovery after dmog washout ( figures s2j  s2m ) . immunostaining showed that hypoxia and dmog reduced synaptic density , as assessed by the decreased co - localization of the presynaptic marker vglut1 and the postsynaptic marker psd-95 ( figures 2e2h ) , or the density of the presynaptic marker synaptophysin ( figures s3p  s3s ) . however , no differences in the abundance or ( plasma membrane ) localization of  the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa ) receptors glua1 and glua2 were detected ( data not shown ) , indicating that the reduced excitatory signaling upon hypoxia or dmog was not due to downregulation of postsynaptic ampa receptors . instead , the reduced synaptic transmission upon hypoxia or dmog results from the remodeling of dendritic spines and the decreased synaptic density . in situ hybridization , rt - pcr , and rna sequencing ( rna - seq ) revealed that phd1 ( also known as egln2 ) and phd2 ( egln1 ) , but not phd3 ( egln3 ) , were detected in the brain of mouse embryos or neonates ( figures s4a  s4c ) . in the adult brain , phd2 was detected in the hippocampus , cortical layers ii , iii , and v , and other regions ( figure  s4a ; data not shown ) . we prepared postsynaptic density ( psd ) fractions from 2-week - old wild - type ( wt ) mice ( cho et  al . , 1992 ) . protein enrichment was assessed by immunoblotting ( ib ) for psd-95 and synaptophysin as post- and pre - synaptic markers , respectively . ib showed that phd2 was broadly present , including in synaptosomes and psds ( figure  3a ) . using an  antibody specific for phd2 in immunostainings ( figures s4d  s4h ) , phd2 was detected in neurites and dendritic protrusions of mhns ( figure  3b ) and rat hippocampal neurons ( rhns ) ( data not shown ) , in close apposition to synaptophysin puncta , suggesting postsynaptic localization . a wide distribution was also obtained in mhns transfected with yfp - tagged phd2 ( phd2yfp ) ( figure  s4i ) . to assess whether phd2 regulated spine formation , we silenced phd2 in mhns . 7-div mhns were co - transfected with yfp and control scrambled ( scr ) short hairpin rna ( shrna ) or phd2-specific shrna ( shphd2 ) . at 14 div , phd2 silencing reduced dendritic protrusion density and the frequency of spines with a head , whereas it increased protrusion length ( figures 3c , 3d , and 3g3i ) . hypoxia did not further alter these parameters in  phd2-silenced mhns ( figures 3e3i ) . time - lapse imaging showed that scr mhns behaved as the control and retained most spines ( figures s4j  s4l , compare with figures s2f s2i , ctrl ) , whereas shphd2 resembled dmog - treated neurons with lower spine density and longer spines ( figures s4j  s4l ) . phd2 silencing did not alter passive and excitable electrical membrane properties , as recorded by patch - clamping , but reduced the frequency of the spontaneously fired aps ( figures s5a  s5j ) . phd2 silencing also impaired synaptic density , as revealed by the reduced number of vglut1/psd-95 co - clusters ( figures 3j3l ) and synaptophysin clusters ( figures s4m  s4q ) . silencing of phd1 did not affect spines ( figures s4r thus , phd2 regulated the formation and maintenance of spines and synapses . to confirm the shrna results , we isolated mhns from phd2 embryos and transfected them at 7 div with the brainbow1.0 ( bbw ) plasmid alone or together with a cre recombinase ( cre ) plasmid . bbw expresses red fluorescent protein ( rfp ) in the absence of cre and cfp or yfp in the presence of cre . compared with control rfp neurons , yfp or cfp phd2-deficient cells had reduced protrusion density and increased protrusion length ( figure  s4u ) . similar results were obtained in mnhs from mice lacking phd2 in neurons ( phd2 ) ( figures 3m3q ; figure  s4v ) . also , phd2 deletion reduced the frequency and synchronicity of spontaneous bursts of aps ( figure  3r ; figure  s5k ) . the phenotype of phd2 spines was rescued by phd2yfp but not by an inactive phd2 mutant that lacks hydroxylation activity ( phd2yfp ) ( vogel et  al . , 2010 ; figure  3s ; figures s4w thus , phd2-mediated hydroxylation is required to induce dendritic spine maturation . to evaluate the effect of phd2 silencing on dendritic spines in  vivo , we performed shrna knockdown of phd2 by in utero electroporation of the hippocampus in embryonic day 15.5 ( e15.5 ) embryos ( pacary et  al . , 2012 ) with a bigenic vector co - expressing the shrna and zsgreen1 fluorescent protein . analysis of dendritic spines in the hippocampal ca1 region on postnatal day 15 ( p15 ) revealed that spine density was reduced in shphd2 neurons in  vivo ( figure  3 t ; figures s6a and s6b ) . golgi staining showed that spine density was reduced in phd2 mice ( figures s6c  s6e ) . to gain insight into the role of phd2 in synapse formation in  vivo transmission electron microscopy ( tem ) and immunostaining revealed that phd2 littermates had reduced synaptic density ( figures s6f and s6 g ) ( number of synapses/100  m , 38.98  0.90 for control ( ctrl ) versus 32.95  1.17 for phd2 ; n  = 3 animals ; p  < 0.001 ) and synaptophysin and psd-95 signals ( figures s6h thus , phd2 regulates spine morphogenesis and synaptic density in  vivo . to identify the molecular mechanism by which phd2-mediated hydroxylation regulated spine morphogenesis , we explored the involvement of hif-1. we evaluated the kinetics of hif - mediated transcription after phd blockade by using the 9xhre::luciferase reporter . luciferase ( luc ) activity was increased , but only after 20  hr of hypoxia or dmog , and the blocker acriflavine abolished this effect ( figure  s1p ) . thus , hif - mediated transcription occurred later than the onset of spine remodeling , suggesting an hif - independent mechanism . also , hypoxia still induced dendritic spine changes in hif-1 mhns ( obtained by transducing mhns from hif-1 mice with an adenoviral cre vector ; data not shown ) , further indicating that spine remodeling occurred independently of hif-1. we considered alternative targets for phd2 and focused on the actin cross - linker flna because it interacts with vhl ( tsuchiya et  al . , 1996 ) , thus providing a putative molecular link between the cytoskeleton and hypoxia . flna is detected in the soma and dendrites , although its localization in spines is debated ( nestor et  al . staining of rhns with a specific anti - flna antibody ( figures s7a and s7b ) used in conjunction with confocal imaging confirmed that flna was detectable in the soma and dendrites and in structures resembling dendritic spines , close to synaptophysin clusters ( data not shown ) . to enhance spatial resolution of flna puncta , we used high - resolution structured illumination microscopy ( sim ) , which showed flna puncta in filopodium - like protrusions and the head of mature dendritic spines ( figure  4a ; figures s7c and s7d ) . the use of sim and yfp - transfected neurons at 14 div , when spines are still remodeling , may explain the more prominent detection of flna in spines compared with other studies using conventional resolution microscopy of untransfected neurons ( which makes precise spine visualization more challenging ) at 34  weeks in culture , when spines and synapses are mature and stable ( noam et  al . , 2012 , nwabuisi - heath et  al . , 2012 ) . to confirm the localization of flna , we transfected mhns with turquoise2 ( tq2)-tagged flna ( flnatq2 ) . in normoxia , flnatq2 was detected in the somato - dendritic compartment , especially in the heads of mature dendritic spines ( figure  4b ) . in hypoxia , flnatq2 was more widespread in the entire neuron , including in filopodia ( figure  4b ) . the c terminus part of flna ( flnatq2 ) , which does not bind actin , showed widespread distribution in mhns independent of oxygen levels ( figure  s7e ) . to provide biochemical confirmation flna was detected in all fractions , notably in the psds ( figure  4c ; figure  s7f ) . exposure of mice to hypoxia ( 8% o2 ) for 4  hr ( a procedure upregulating hif target genes ; figure  s7 g ) altered flna levels and/or distribution ( figure  4c ; figure  s7f ) . flna was especially increased in the non - synaptosomal membrane fraction ( figure  4c ; figure  s7f ) . thus , flna was detectable in dendritic spines and psds of neurons , and its localization and levels were influenced by hypoxia . time - course studies in mhns revealed that flna protein levels increased already within 15  min ( for dmog ; data not shown ) and remained elevated for several hours ( figures 4d and 4e ; figures s7h and s7i ) . this upregulation was independent of gene transcription or protein translation because flna transcript levels were not induced during the first 4  hr ( figure  s7j ) , and treatment of hypoxic mhns with actinomycin d or cycloheximide did not prevent flna protein upregulation ( figure  4f ; figure  s7k ) . however , flna was upregulated when normoxic mhns were treated with the proteasomal inhibitor mg132 in a dose - dependent manner ( figures 4f and 4 g ; figures s7k and s7l ) . the combination of hypoxia plus mg132 did not further elevate flna levels above those in hypoxia ( data not shown ) , suggesting that hypoxia and proteasomal inhibition act via the same mechanism . ib revealed that phd2 silencing increased flna levels ( figure  4h ; figure  s7 m ) . flna levels were also higher in homogenates and non - synaptosomal fractions of phd2 brains ( figure  4i ; figures s7n s7s ) . thus , oxygenation and phd2 regulated flna protein levels via a mechanism involving proteasomal degradation . prompted by the similarities between the regulation of flna and hif-1 by the phd2/proteasome axis ( epstein et  al . , 2001 ) , we hypothesized that flna was a target of phd2-mediated proline hydroxylation and that hydroxylated flna underwent ubiquitination and proteasomal degradation . we co - transfected hek293 t cells  with myc - tagged flna ( mycflna ) and phd2yfp or phd2yfp . co - immunoprecipitation ( coip ) showed that phd2yfp and phd2yfp interacted with mycflna ( figure  5a ) . flna and phd2 also formed a complex in brain psds but not in the non - synaptic fraction ( figure  5b ; figure  s8a ) . flna contains an actin binding domain ( abd ) , 24 immunoglobulin g ( igg ) domains ( d1d24 ) , and two flexible regions ( hinges h1 and h2 ) ( stossel et  al . , 2001 , zhou et  al . , 2010 ) to analyze which domains of flna interacted with phd2 , we generated five myc - tagged deletion variants of flna lacking domains known to interact with different binding partners ( zhou et  al . proper folding of the flna variants was confirmed because mycflna and mycflna mutants bound to f - actin ( data not shown ) , and mycflna and mycflna mutants ( comprising the d24 dimerization domain ) interacted with endogenous flna ( figure  s8b ) . coips showed that phd2yfp and phd2yfp interacted with the deletion mutants mycflna and mycflna but not with other domains ( figure  5c ; figure  s8c ; data not shown ) . complex detection was enhanced when using phd2yfp , suggesting that they were stabilized in the absence of hydroxylation ( figure  s8c ) . we confirmed the phd2-flna interaction by using fluorescence lifetime imaging microscopy ( flim ) to map the fluorescence resonance energy transfer ( fret ) between the donor flnatq2 and the acceptor phd2-yfp ( using yfp as a control ) . co - expression of flnatq2 and yfp did not change the fluorescence lifetime of the donor ( figure  5d ) . in contrast , co - expression of flnatq2 and phd2yfp increased fret , as evidenced by the reduced tq2 fluorescence lifetime ( figure  5d ) , indicating that they formed a complex in the range  of 110  nm . similar results were obtained when using phd2yfp ( figure  5d ) . thus , amino acid residues 22502508 in flna were necessary for flna - phd2 binding , but the hydroxylation activity of phd2 per se was not required . although phds hydroxylate prolines in hif-1 by recognizing an lxxlap motif , other phd targets lack such a motif ( wong et  al . , the mycflna fragment contains 20 prolines but none in an lxxlap motif ( figure  s8d ) . we examined the ability of each proline to interact with phd2 by generating glutathione s - transferase ( gst ) fusion peptides , each containing a proline together with six up- and downstream flanking residues ( song et  al . , 2013 ) . ib of precipitated complexes revealed that only peptides containing the prolines p2270 , p2309 , p2316 , p2320 , p2404 , and p2439 interacted with phd2 ( figure  s8e ) . a weaker interaction was observed with peptides containing p2294 and p2312 . to analyze whether phd2 hydroxylated flna prolines , we used an antibody specific for hydroxylated prolines ( oh - pro ) . hek293 t cells were transfected with mycflna and treated with the proteasomal inhibitors mg132 or lactacystin , showing that proteasome inhibition increased flna hydroxylation ( figure  5e ; figure  s8f ) . flna was also hydroxylated in  vivo , and hydroxylation was reduced in rats exposed to hypoxia ( 8% o2 ; figure  5f ) . to identify which prolines were hydroxylated by phd2 , we  transfected hek293 t cells with plasmids encoding mycflna alone or together with phd2yfp . cells were treated with vehicle or mg132 , and immunoprecipitated mycflna was digested for liquid chromatography - mass spectrometry ( lc - ms ) . hydroxylation of residues p2309 , p2312 , and p2316 in fneehipdspfvvpvaspsgdar ( p : oh - pro ) were enhanced in double - transfected cells . the probability of hydroxylation was p2316  > p2309  > p2312 , identifying p2316 and p2309 as the most probable phd2 hydroxylation sites ( figure  5 g ; figure  s8 g ; data not shown ) . we also mutated p2309 and p2316 to alanine ( mycflna , mycflna ) and tested their binding to phd2 and hydroxylation upon co - transfection with phd2yfp . although both sequence mutants interacted with phd2yfp , they were less hydroxylated than mycflna ( figure  5h ; figure  s8h ) . moreover , in the presence of dmog , hydroxylation of mycflna was reduced to levels obtained with both sequence mutants ( figure  5h ; figure vhl ubiquitinates hydroxylated hif to mark it for proteasomal degradation ( ivan et  al . , 2001 , jaakkola et  al . , 2001 ) . because flna interacts with vhl ( tsuchiya et  al . , 1996 ) , and vhl was present in the psds ( figure  4c ) , we explored whether vhl mediated flna ubiquitination . we first validated the interaction between vhl and flna in mg132-treated hek293 t cells cotransfected with ha - tagged vhl ( ha - vhl ) and mycflna ( figure  6a ) . flna deletion mutants showed that vhl bound to mycflna and mycflna ( figure  6b ; data not shown for mycflna and mycflna ) . the single mutants mycflna and mycflna were still able to interact with vhl , whereas the triple mutant p2309/2312/2316a ( mycflna ) showed a weaker interaction ( figure  6c ; figure  s9a ) . taken together , our results  suggest that hydroxylation of p2309 , p2316 , and possibly p2312 was required for the interaction of flna with  vhl . transduction of mhns with a lentivirus expressing scr or vhl - specific shrna ( shvhl , reducing vhl levels by 67%  4% ; n  = 3 ; p  < 0.05 ) showed higher flna protein levels in vhl - silenced neurons ( figure  6d ) . to confirm that vhl regulates flna levels in  vivo , we generated vhl mice by crossing vhl mice with nestincre mice . because these mice are lethal at e16.5 , we made brain homogenates from e14.5 control or vhl littermates . as expected , hif-1 levels were increased in vhl embryos ( figure  6e ) . notably , flna levels were higher in vhl mice ( figure  6e ; figure  s9b ) . because protein abundance in e14.5 embryo brains was insufficient for ip , e14.5 vhl brain extracts were blotted for oh - pro and reblotted thereafter for flna . this analysis showed that the increased flna levels were comparably hydroxylated in vhl mice ( figure  6f ; figures s9c s9e ) , suggesting that proline - hydroxylated flna was stabilized in the absence of vhl in  vivo . we then assessed whether the interaction of hydroxylated flna with vhl resulted in flna ubiquitination . brains from e14.5 vhl and control littermates were collected in the presence of mg132 and pr-619 . tandem ubiquitin binding entities ( tube ) pull - down of poly - ubiquitinated proteins followed by ib showed that flna was ubiquitinated less in vhl brains ( figure  6 g ; figure  s9f ) . we assessed whether flna stabilization was responsible for the  remodeling of dendritic spines to a more immature state . because forced overexpression of full - length flna was toxic ( data not shown ) , we used another strategy to elevate endogenous flna to physiological levels . we took advantage of the  observation that overexpression of mycflna increased the levels of endogenous flna 2-fold ( figures 4h and 7a ; figure  s7 m ) , akin to the effect of hypoxia , dmog , or phd2 silencing ( figures 4d4f , 4h , and 7a ; figures s7h , s7i , s7k , and s7 m ) . flna elevated flna levels by impairing the interaction between endogenous flna and phd2 because less phd2 was coimmunoprecipitated with mycflna in the presence of gst - flna ( figure  7b ) . we therefore tested whether mycflna expression , and concomitant elevated flna levels , impaired dendritic spine maturation . mhns transfected with mycflna had fewer and less mature dendritic spines ( figures 7c , 7d , and 7f7h ) and formed fewer synaptic contacts ( figures 7i7l ) , comparable with the response to hypoxia ( hypoxia versus mycflna , p > 0.05 for all parameters ; data not shown ) . because flna also interacts with rac and cdc42 , which promote mature spine formation ( nakamura et  al . , 2011 , tashiro and yuste , 2004 ) , a possible dominant - negative interaction of  mycflna with these small gtpases might also  contribute to the more immature spine phenotype of mycflna - expressing neurons . to assess the specificity of this peptide in mediating the phd2-regulated flna degradation notably , this mutant failed to impair spine maturation and synaptic contact formation ( figures 7e7h , 7k , and 7l ) . although we can not formally exclude that mycflna perturbs the interaction with other binding partners of flna , the lack of an effect by mycflna strongly suggests that the effect of mycflna on spine morphology was due to its interaction with phd2/vhl and to flna degradation rather than to non - specific dominant - negative interactions with other proteins . notably , 14-div mhns of e15.5 vhl mice ( containing higher levels of endogenous flna ) also showed more immature dendritic spines ( figures s9 g and s9h ) . we co - transfected mhns with tdt together with scr or flna - specific shrna ( shflna ) alone or together with shphd2 . shflna reduced flna protein  levels by 59%  8% ( n  = 3 , p  < 0.05 ) . silencing of flna in mhns alone increased protrusion length without affecting the protrusion density or the number of spines with a head ( figures 7m7s ) . however , in phd2-silenced mhns , flna silencing largely prevented the switch to a more immature spine phenotype ( figures 7m7s ) . our study shows that oxygen , via phd2 , regulates dendritic spines and synaptic transmission . phd2 coordinates rapid , reversible adaptations of spines , at least in part , by controlling flna levels . when oxygen is limited , phd2 is inactivated , which diminishes proteasomal degradation of flna , thus inducing its stabilization and accumulation in non - synaptic locations and increasing the ratio of flna to f - actin . based on reports in other cells ( van der flier and sonnenberg , 2001 ) , we speculate that the increased flna abundance results in remodeling of the actin cytoskeleton from a mesh network in mature spine heads to parallel f - actin bundles in headless immature elongated protrusions . our data suggest that diminished degradation of flna upon  phd2 inactivation in hypoxia rearranges the actin cytoskeleton to reduce the number of dendritic spines , synapses , and synaptic transmission without affecting intrinsic electrical membrane properties . our findings are in line with a report showing that chemical inhibition of phds and phd2 deficiency reduce long - term potentiation ( corcoran et  al . , 2013 ) . experiments targeting flna to dendritic spines by using a spine - targeting domain ( such as of sg2na ; gaillard et  al . , 2006 ) to inhibit phd2 locally in the spines could be designed to confirm the model that phd2 activity , locally at the spines , mediates spine maturation . phds orchestrate the levels of target proteins via hydroxylation of prolines , although hydroxylation - independent activities have also been reported ( wong et  al . , 2013 ) . using a combination of biochemical , imaging , and site - directed mutagenesis methods , we show that phd2 interacts with domains d21d23 of flna and hydroxylates p2309 and p2316 ( conserved in zebrafish , mouse , rat , and human ) , favoring its ubiquitination and proteasomal degradation . inactivation of phd2 hydroxylation activity or mutation of p2309 and p2316 to alanine impaired flna hydroxylation . genetic studies in  vitro and in  vivo confirmed the importance of phd2 and vhl in dendritic spine remodeling . together , these studies identify flna as a target of phd2 involved in the formation and maturation of dendritic spines . in immortalized cell lines , hypoxia induces flna cleavage ( zheng et  al . , 2014 ) . however , hypoxia or dmog did not upregulate such an flna fragment in mhns ( data not shown ) , perhaps because the effects of hypoxia on flna levels are cell - type dependent or differ upon immortalization . in any case , flna protein levels in primary mhns were increased  not decreased  upon exposure to hypoxia or dmog . although spine remodeling by phd2 is hif - independent , we can not exclude that other phd2 targets , apart from flna , influence spine remodeling . somewhat counterintuitively , both the modest elevation of flna ( by mycflna ) and silencing of flna resulted in similar  although not exactly identical  spine changes . indeed , silencing of flna increased protrusion length without affecting the protrusion density or the number of spines with a head , whereas mhns transfected with mycflna had fewer and less mature dendritic spines . this apparent contradiction likely relates to the well - known fact that flna interacts with several partners and has contextual effects and that flna protein levels require precise regulation . indeed , both under- and overexpression of flna impair neuronal migration , although via distinct molecular mechanisms ( sarkisian et  al . , 2006 , zhang et  al . , 2012 , zhang et  al . , 2013 ) . furthermore , both loss and gain of flna / cheerio in humans and flies cause similar neurological defects , although distinct symptoms have also been reported ( bolduc et  al . , 2010 , zhou et  al . , 2010 ) . our findings that flna is post - translationally regulated by phd2-mediated hydroxylation and vhl - mediated ubiquitination unravel a molecular mechanism for the precise regulation of flna abundance , required for the proper formation and maintenance of synapses . our findings that hypoxia rapidly induces reversible spine regression may help to better understand how neurons adapt to a hypoxia challenge . because synaptic transmission is a high - energy - consuming process and neurons rely on oxygen to produce atp , one possible mechanism whereby neurons can avoid an energy crisis is by decreasing synaptic transmission through spine regression . importantly , this remodeling is not irreversible ( which would otherwise impair brain performance permanently ) , but phd2/flna may provide a molecular mechanism for reversible spine maturation and re - establishment of synapses upon return to sufficient oxygen conditions . although our study was not designed to provide direct proof for such a protective mechanism , previous reports support this hypothesis . first , when atp production is compromised , dendritic arborization is impaired ( oruganty - das et  al . , 2012 ) . second , both an increase in spine length and a rapid reversible spine retraction protect against excitotoxic damage in hypoxia ( araya et  al . third , in hypoperfused peri - infarct regions , neurons lower the number of mature spines and increase the length of protrusions to protect against excitotoxic overstimulation and to rewire new functional circuits ( brown et  al . , 2010 ) . we used swiss , phd2 , phd2 , nestincre , vhl , and hif-1 mice and wistar rats . in utero electroporations hippocampal neurons were isolated and cultured as described previously ( segura et  al . , 2007 ) . hypoxic incubations were at 0.5%1% ( neurons ) or 0.2% o2 ( hek293 t ) . transfections were done by lipofectamine 2000 ( neurons ) , calcium phosphate ( hek293 t ) , or nucleofection ( pc12 ) . rna expression analysis was done by in  situ hybridization , rt - pcr , or rna - seq . immunostaining was performed on 4% paraformaldehyde ( pfa)-fixed hippocampal neurons or thick free - floating brain cryo- or vibratome sections . t cells transfected with mycflna with or without phd2yfp were incubated with vehicle or 10  m mg132 for 2  hr before lysis . cell lysates were immunoprecipitated with anti - myc antibodies and fractionated by sds - page . the section containing mycflna was trypsinized in gel , and digested peptides were analyzed by nano - lc - ms / ms . bright - field , fluorescent ( time - lapse ) confocal imaging , super - resolution imaging ( sr - sim ) , tem , and time - domain flim were performed as described in the supplemental experimental procedures . patch - clamp recordings in mhns were performed as described previously ( reinartz et  al . , 2014 ) . extracellular recordings of the spontaneous network - level electrical activity , arising among neurons growing over multi - electrode arrays ( meas ) , were performed as reported previously ( gambazzi et  al . , statistical differences were calculated by two - tailed unpaired t  test for two datasets or anova followed by bonferroni post hoc test for multiple datasets using prism ( graphpad ) . i.s . , c.l . , m.d . , and p.c . wrote the manuscript .
summaryneuronal function is highly sensitive to changes in oxygen levels , but how hypoxia affects dendritic spine formation and synaptogenesis is unknown . here 
 we report that hypoxia , chemical inhibition of the oxygen - sensing prolyl hydroxylase domain proteins ( phds ) , and silencing of phd2 induce immature filopodium - like dendritic protrusions , promote spine regression , reduce synaptic density , and decrease the frequency of spontaneous action potentials independently of hif signaling . 
 we identified the actin cross - linker filamin a ( flna ) as a target of phd2 mediating these effects . in normoxia , phd2 hydroxylates the proline residues p2309 and p2316 in flna , leading to von hippel - lindau ( vhl)-mediated ubiquitination and proteasomal degradation . in hypoxia , 
 phd2 inactivation rapidly upregulates flna protein levels because of blockage of its proteasomal degradation . 
 flna upregulation induces more immature spines , whereas flna silencing rescues the immature spine phenotype induced by phd2 inhibition .