parkinson 's disease ( pd ) is characterized by the formation of neuronal inclusion bodies that are denoted as lewy bodies . the -synuclein protein is a presynaptic and its function and involvement in the development of pd is yet to be clearly determined . when -synuclein is abnormally expressed or modified , various studies indicate it may cause alterations in mitochondrial and proteasomal function , protein aggregation , and accumulation of reactive oxygen species ( ros ) [ 36 ] . increased ros and cellular -synuclein levels promote its aggregation [ 7 , 8 ] and can cause posttranslational modifications to the methionine ( met ) residues of -synuclein leading to the formation of methionine sulfoxide ( meto)containing -synuclein ( meto--synuclein ) . meto modifications can be present in two forms of enantiomers : met - s - o and met - r - o that can be readily reduced by the methionine sulfoxide reductase ( msr ) system . msr type a ( msra ) reduces met - s - o and msr type b ( msrb ) reduces met - r - o [ 9 , 10 ] . msra is thought to be the major msr because it is a positive regulator of msrb expression levels [ 11 , 12 ] . absence of msra can cause a hypervulnerability to conditions of oxidative stress [ 1317 ] and shortened lifespan [ 16 , 18 ] . one possible explanation is that the cellular regulation of aging neurons ( e.g. , in pd ) is altered and consequently causes the accumulation of misfolded proteins ( e.g. , -synuclein ) . previous work has determined that msra knockout yeast strain expressing either of the three -synuclein types ( normal type -synuclein ( syn ) and syn mutant types : syna30p , syna53 t ) showed significantly lower levels of phosphorylation relative to -synuclein expressed in wild - type cells . these data suggest that increased levels of meto moiety in -synuclein could inhibit its phosphorylation efficiency , especially when expressed in msra - ko cells . supportive evidence for such possibility was demonstrated by a reduced in vitro phosphorylation of these three types of -synuclein by casein kinase 2 ( a major kinase for -synuclein ser phosphorylation ) following oxidation of -synuclein . the observed decrease in -synuclein phosphorylation after meto formation when msra is ablated in yeast cells may mimic processes that regulate -synuclein phosphorylation in mammals . further investigations in a parallel mammalian system will greatly contribute to clarify the physiological role of msra on -synuclein phosphorylation . accordingly , the goal of the current studies is to determine the levels of meto and -synuclein phosphorylation in brains of msra knockout ( msra ) and wt mice and evaluate their intracorrelation . [ -p]-atp ( 3,000  ci / mmol ) was purchased from perkin - elmer ( waltham , ma ) and casein kinase 2 was purchased from millipore ( billerica , ma ) . rabbit anti - meto antibodies were made in our lab   and antibodies against -synuclein were purchased from bd . protein extractionpostmortem brains of each mouse types ( 6 months of age ) were homogenized at 4c in the presence of 25  mm tris ( ph 7.4 ) and protease inhibitor cocktail ( no - edta ) ( roche , south san francisco , ca ) and 1  mm cacl2 . the extracted protein fractions were centrifuged ( 15,000  g ) for 20 minutes at 4c , and the resulting tris - soluble supernatants ( sup 1 ) were kept and stored at 80c . the pellets obtained following the centrifugation step were redissolved in 25  mm tris ( ph 7.4 ) and 48% urea . then , the urea - soluble pellets were centrifuged again as described above , and the resulting supernatants were dialyzed against 25  mm tris ( ph 7.4 ) at 4c ( sup 2 ) . postmortem brains of each mouse types ( 6 months of age ) were homogenized at 4c in the presence of 25  mm tris ( ph 7.4 ) and protease inhibitor cocktail ( no - edta ) ( roche , south san francisco , ca ) and 1  mm cacl2 . the extracted protein fractions were centrifuged ( 15,000  g ) for 20 minutes at 4c , and the resulting tris - soluble supernatants ( sup 1 ) were kept and stored at 80c . the pellets obtained following the centrifugation step were redissolved in 25  mm tris ( ph 7.4 ) and 48% urea . then , the urea - soluble pellets were centrifuged again as described above , and the resulting supernatants were dialyzed against 25  mm tris ( ph 7.4 ) at 4c ( sup 2 ) . fifty microgram of sup 1 and a mixture containing 40  g of sup 2 and 10  g of sup 1 ( serving as a source for kinases ) were incubated in the presence of 25  mm tris ( ph 7.4 ) , protease inhibitor cocktail ( no - edta ) ( roche , south san francisco , ca ) , 1  mm cacl2 , 10  mm mgcl2 , and 16.7  m [ -p]-atp ( 3000 ci/ mmol ) for 3 minutes at room temperature in a final volume of 50  l . endogenous phosphorylation was stopped by addition of 10  mm edta , 10  mm egta , and 1  mm cold atp and immediately placed on ice . then , the samples were subjected to an immunoprecipitation by anti--synuclein antibodies or anti - meto antibodies as described below . postmortem brains ( n = 5 ) of each mouse types were processed according to the protocol provided by the phosphoprotein purification kit purchased from qiagen ( valencia , ca ) . briefly , each brain tissue was homogenized using 0.25% ( w / v ) chaps solution in lysis buffer containing protease inhibitor cocktail and benzonase . following incubation at 4c for 30  min , homogenates were centrifuged at 10,000    g and 4c for 30  min . protein concentrations of the corresponding supernatants were determined by coomassie ( bradford ) protein assay kit ( thermo fisher scientific , rockford , il ) , and 2.5  mg of total protein of each lysate was applied into the phosphoprotein purification column according to the protocol instructions . then , the unbound proteins were washed out from the column and the column - bound proteins were eluted with the elution buffer and concentrated using centricon with molecular cutoff of 3  kda ( millipore , billerica , ma ) . finally , equal protein amounts ( 50  g ) of each concentrated sample were subjected to an sds - page ( 4%20% ) separation followed by western blot analysis using primary antibodies against -synuclein ( bd , franklin lakes , nj ) . immunoprecipitation was performed on brain soluble fractions described above ( sup1 and sup2 ) by using primary antibodies against either -synuclein ( bd , franklin lakes , nj ) or antibodies against meto . the immunoprecipitation procedure was followed according to the immunoprecipitation kit 's protocol and related supplies provided by pierce inc . thereafter , equal protein amounts of the immunopercipitants were subjected to an sds - gel electrophoresis ( 4%20% ) followed by either western blot analysis using anti--synuclein antibodies ( as primary antibodies ) or directly exposed to x - ray film when [ -p]-atp was used in the phosphorylation assay . the levels of the resulting reacting bands were quantified by densitometry analysis . in the western blot analysis using the primary anti--synuclein antibodies , normalization of the -synuclein detected bands in the meto precipitants was normalized to the levels of -synuclein bands detected directly in each protein extract fraction prior to the immunoprecipitation procedure ( 50  g protein per lane ) . previously , we have showed that met oxidation inhibits the phosphorylation of -synuclein both in vitro and in ex vivo systems , in which -synuclein was overexpressed in yeast . in addition to the phosphorylation inhibition of -synuclein expressed in yeast , general protein degradation and -synuclein degradation were inhibited as well in this ex vivo system while increasing protein fibrillation . to establish the relationships between -synuclein phosphorylation and methionine oxidation in a mammalian model , we have monitored first the levels of methionine - oxidized -synuclein in msra   compared with control wild - type mice . accordingly , the levels of total -synuclein and meto--synuclein were monitored in wt and msra   brains by western blot analysis , following immunoprecipitation by the novel anti - meto antibodies ( figure 1(a ) ) . the presented data clearly show that in comparison to wt , the levels of meto -synuclein are significantly elevated in msra   brains both within the tris - soluble ( sup 1 ) and urea - soluble ( sup 2 ) fractions as demonstrated by band densitometry analysis ( figure 1(b ) ) . these results confirm our initial observation showing that the levels of meto - containing proteins are increased in msra   brain   and in serum proteins as function of age . in addition , the percent of meto in the analyzed fractions is similar to the in vivo meto percentile observed in yeast cells exposed to oxidative stress [ 14 , 15 ] ( the percent meto values were calculated as percent ratio of the immunoprecipitated meto--synuclein bands ( figure 1(a ) , lanes 58 ) to the corresponding bands representing total -synuclein levels ( figure 1(a ) , lanes 14 ) ) . the next step was to evaluate the effect of the observed methionine oxidation of -synuclein on its phosphorylation levels in brain . to mimic in vivo phosphorylation , brain fractions were incubated in a reaction mixture containing [ p]-atp followed by immunoprecipitation with anti--synuclein antibodies or anti - meto antibodies . then , the resulting precipitates were subjected to sds - page and levels of radiolabeled proteins were detected and quantified by exposure to x - ray film . if indeed methionine oxidation interferes with protein phosphorylation , it is expected that levels of phosphorylation detected in the precipitated proteins will be in inverse correlation with the levels of meto in these proteins . as shown in figure 2(a ) , immunoprecipitation of -synuclein resulted in significantly higher levels of phosphorylated -synuclein in the wt 's urea - soluble fraction compared with the corresponding msra   fraction . interestingly , phosphorylated -synuclein was detected only in the urea - soluble fraction and not detected in the tris - soluble fraction ( figure 2(a ) , lanes 2 and 4 ) . these results confirm our initial observation in the ex vivo system , in which the phosphorylation of recombinant -synuclein was inhibited in msra null mutant yeast cells . moreover , the demonstrated enhanced protein fibrillation in -synuclein - expressing msra knockout yeast cells may relate to the lower -synuclein phosphorylation in membranal / insoluble msra   fraction , as shown here in mouse brain ( figure 2(a ) ) . the role of phosphorylation of -synuclein is controversial . in vitro phosphorylation of -synuclein at ser by casein kinase 2 forms fibrils faster than nonphosphorylated -synuclein   and fosters the formation of cytoplasmic inclusion bodies in cell culture models of synucleinopathies however , in vivo studies showed no obvious correlation between phosphorylated ser and -synuclein fibril formation . in contrast , another study demonstrated that phosphorylation at ser inhibits -synuclein fibril formation in vitro . in light of the recent and current data , it is intriguing to propose that phosphorylation of -synuclein may be a cellular compensatory attempt aimed at increasing its solubility and preventing its aggregation . this suggested role of -synuclein phopshorylation is especially important under conditions of oxidative stress that lead to enhanced methionine oxidation . typically , protein oxidation will cause a met residue to be more hydrophilic , but the overall meto protein may become more hydrophobic by exposing hidden hydrophobic residues . therefore , enhanced formation of meto--synuclein ( while causing its reduced phosphorylation rate ) is expected to increase its hydrophobic properties and as a consequence of its aggregation . immunoprecipitation of [ -p]-labeled proteins by anti - meto antibodies revealed a distinct protein band with a similar molecular mass of -synuclein ( figure 2(a ) , lanes 6 and 8) . to facilitate discussion , this protein band will be denoted as meto-15 . like shown with the [ -p]-labeled -synuclein precipitants ( figure 2(a ) ) , the relative intensity of this band was greater in the wt 's urea - soluble fraction than its parallel msra   fraction . although the wt fraction contains meto proteins ( as evident by the performed meto - protein immunoprecipitation ) , the meto residue levels in the wt fraction are lower compared with msra   fraction , and thereby causing higher phosphorylation level of the detected wt 's meto-15 ( figure 2(a ) ) . the identification of this meto-15 band by mass spectrometry was not possible due to limits of detection . however , by its molecular mass , it seems that this protein may be either meto--synuclein or a protein with a similar molecular mass as previously observed . regardless of meto-15 identity , the phosphorylation levels of this protein provide a good indication on the meto effect on protein phosphorylation in each mouse type . to better relate to the levels of brain -synuclein phosphorylation in each mouse type , densitometry analyses of the phosphorylated -synuclein bands were performed . the averaged value calculated for the wt brain represented a 100% phosphorylation rate ( figure 2(b ) ) . accordingly , the averaged level of phosphorylated -synuclein in the msra   urea - soluble fraction was inhibited by 60% in comparison to the wt 's urea - soluble fraction ( figure 2(b ) ) . this meto - mediated inhibition of -synuclein phosphorylation through msra gene ablation is in agreement with the presence of significantly higher levels of in vivo methionine oxidation of -synuclein in msra   brain ( figure 1 ) . additional supportive evidence for this phenomenon is the observation showing that in vivo phosphorylation of -synuclein is lower by 36% in msra   compared with wt brain ( figure 2(c ) ) . it is suggested that a compromised msra function may cause alteration of -synuclein 's methionine sulfoxide levels that in turn affect -synuclein 's phosphorylation levels . consequently , the ratio of -synuclein 's phosphorylated form to its nonphosphorylated form may contribute to the progression of neurodegenerative diseases like pd . other in vitro effects of methionine oxidation on -synuclein have been extensively described in the literature . for example , the role of methionine oxidation on -synuclein fibrillation has been studied in vitro , and it seems that meto inhibits -synuclein fibrillation , depending on the environmental conditions [ 3035 ] . in addition , it was reported that meto may affect dopamine binding to -synuclein [ 36 , 37 ] and its ability to carry out one of its proposed functions as an antioxidant .   in summary , it is possible that -synuclein structure and function may be regulated through in vivo oxidation of its met residues and their reversal reduction by the msr system . further investigations are underway to determine if the observed in vitro effects of meto on -synuclein 's structure and function can also be detected in vivo .
previously , we have showed that overexpression of methionine - oxidized -synuclein in methionine sulfoxide reductase a ( msra ) null mutant yeast cells inhibits -synuclein phosphorylation and increases protein fibrillation . 
 the current studies show that ablation of mouse msra gene caused enhanced methionine oxidation of -synuclein while reducing its own phophorylation levels , especially in the hydrophobic cell - extracted fraction . 
 these data provide supportive evidence that a compromised msra function in mammalian brain may cause enhanced pathologies associated with altered -synuclein oxidation and phosphorylation levels .