-synuclein is a 140 amino acid brain protein , mainly 
 localized in presynaptic terminals [ 1 , 2 ] . although the 
 detailed physiological functions of -synuclein are still elusive , recent studies suggest that it plays 
 a key role in synaptic functions cooperated with cysteine - string 
 protein- ( csp  ) , which contains a typical domain 
 for hsp40-type molecular cochaperones . in the subgroup of neurodegenerative disorders termed synucleinopathies ,  
 -synuclein is known to polymerize into fibrils and to 
 accumulate in pathologic hallmark inclusions , such as lewy body 
 ( lb ) , lewy neuritis ( ln ) , and glial cytoplasmic inclusions ( gcis ) . 
 the lb and ln are characteristic of parkinson 's disease ( pd ) , and 
 point mutations or gene multiplications of -synuclein are 
 responsible for familial pd [ 46 ] . moreover , transgenic 
 flies overexpressing mutated human -synuclein showed 
 progressive locomotor disability with dopaminergic neuronal cell 
 death with intracytoplasmic inclusions . these findings 
 suggest that abnormal -synuclein metabolism plays a key 
 role in neurodegenerative processes in pd and other 
 synucleinopathies , but the precise underlying mechanisms still 
 remain unknown . to elucidate the possible roles of 
 -synuclein in neurodegeneration , we have developed cells 
 that overexpress   wild - type or mutant -synucleins in 
 dopaminergic or inducible catechol - quinone producing cell lines [ 9 , 10 ] . the inclusions in synucleinopathies were proved to be composed of -sheet rich fibrils formed by nitrated species of 
 -synuclein . several lines of evidence suggested that reactive oxygen species ( ros ) play a key role in the 
 conformational change of -synuclein and the following 
 aggregate formation [ 1215 ] . we developed human dopaminergic sh - sy5y cells overexpressing wild - type or mutant -synucleins , and established experimental models of 
 intracellular aggregate formation following the exposure to 
 various ros . the aggregates thus formed were 
 immunopositive for ubiquitin , nitrotyrosine , and dityrosine , and positive for thioflavin s staining , which was in good agreement with the pathological features of 
 inclusion bodies in synucleinopathies . the 
 -tubulin and molecular chaperones coexisted as well , 
 suggesting that the aggregate formation was associated with the 
 intracellular transport system for protein turnover responses 
 against the toxic effects of misfolded proteins . such mechanisms 
 are called  aggresome  and are suggested to represent one of the 
 cytoprotective responses [ 1618 ] . interestingly , the recent study on huntingtin showed that inclusion body formation 
 reduced the risk of neuronal death . however , it is still controversial whether the aggregate formation of 
 -synuclein has cytotoxicity in the neuronal cell or 
 sequesters toxic species . we established a cellular model in which intracellular 
 -synuclein aggregations were efficiently formed in 
 response to various types of ros exposure [ 9 , 19 ] . under these 
 conditions , a significant number of cells showed caspase 3 
 activation . to explore possible relationships between the aggregate formation and   apoptosis , first we investigated 
 whether -synuclein aggregates colocalized with activated 
 caspase-3 using a double immunostaining method . following the 
 combined exposure of the cells to a no donor and rotenone surprisingly , 
 immunocytochemical analyses revealed that the aggregate positive 
 cells did not show any caspase 3 activations and , conversely , that 
 caspase 3 activated cells did not contain any -synuclein aggregates . iron was able to induce -synuclein aggregates more effectively than any other ros inducers 
 and no donors , suggesting the iron plays a key role in the 
 aggregate formation . when using both ros and no inducers , the addition of ferric iron triggered further aggregate formation , 
 but cells positive for activated caspase 3 were not coincident 
 with aggregate positive cells . in quantification experiments , it 
 was revealed that caspase 3-positive cells were decreased by the 
 addition of ferric iron . on the other hand , by chelating ferric 
 iron , the aggregate formation was decreased with concomitant 
 increases of caspase 3 activation . these data suggest that the 
 ferric iron plays a key role in the -synuclein aggregation . furthermore , these data also imply that the aggregate 
 formation may be cytoprotective against various cellular insults 
 including oxidative stress [ 19 , 20 ] . since -synuclein is ubiquitously expressed at 
 high levels in all brain regions , the mechanisms responsible for the preferential and selective neurodegeneration 
 of dopaminergic neurons in the substantia nigra remain to be 
 determined . previous studies suggested that the specific 
 vulnerability of dopaminergic neurons may be linked to the 
 cytotoxic oxidative potential of dopamine . highly reactive oxygen species ( ros ) are generated not only in dopamine 
 oxidation but also during the decay of catechol - derived 
 orthoquinones which covalently incorporate into a variety of 
 molecules including proteins and nucleic acids . on the other hand , previous reports demonstrated that -synuclein 
 might regulate dopamine metabolism by direct interaction with the 
 tyrosine hydroxylase , the dopamine transporter 
 and vesicular monoamine transporter ( vmat2 ) , key proteins in the 
 regulation of the dopamine content within nerve terminals 
 [ 26 , 27 ] . therefore , the pathological metabolism of -synuclein may be closely linked to the misregulation of 
 dopamine , consequently leading to neuronal death . in support of 
 this notion , catechol - derived orthoquinones ( eg , dopamine - quinone 
 or dopa - quinone ) accelerate and stabilize the formation of 
 -synuclein protofibrils by inhibiting the conversion of 
 toxic protofibrils into fibrils [ 28 , 29 ] . to shed light on the pathophysiological mechanisms underlying 
 -synuclein - mediated neurodegeneration in dopamine neurons , 
 we developed novel neuronal cell lines coexpressing 
 -synuclein ( wild - type or a53 t ) and tyrosinase that 
 produces catecholamines and their oxidized metabolites [ 30 , 31 ] . investigating the effects of wild - type or mutant -synuclein expression , we found that the coexpression of 
 wild - type and a53 t mutant -synuclein in tyrosinase - overexpressing cells exacerbated dna damage and 
 successive apoptotic cell death compared to the cells 
 overexpressing cat or antisense -synuclein . both wild - type 
 and a53 t mutant -synucleins coexpressed with tyrosinase 
 resulted in the gradual accumulation of high - molecular weight 
 complexes immunopositive for -synuclein . this band , 
 possibly representing oligomerized forms , corresponded to the size 
 of -synuclein tetramer and was also detected by the 
 nbt / glycinate redox - cycling staining , suggesting that it was 
 modified by quinones . moreover , during these processes , the mitochondrial membrane 
 potential was specifically decreased without the activation of map 
 kinases . although the underlying mechanism(s ) 
 of neuronal cell death following the coexpression of tyrosinase and 
 -synuclein are still elusive , it is likely that 
 -synuclein modified by the oxidized catechol metabolites 
 forms cytotoxic intermediates , that is , recent 
 reports suggested that protofibrillar -synuclein tightly binds to lipid bilayers and 
 increases the membrane permeability by forming pore - like structures 
 [ 3335 ] . while the membranous structures damaged by protofibrils in dopaminergic nerve 
 terminals remain unknown , intracellular organelles , such as synaptic vesicles and mitochondria , 
 are possible candidates . in this regard , disruption of synaptic 
 vesicle membranes would result in an increase of the cytoplasmic 
 dopamine levels that would trigger the further accumulation of 
 dopamine - quinone and dopamine - derived oxyradicals and thus lead to 
 a vicious cycle . likewise , mitochondrial enzymes in the electron 
 transport chain and the functional permeability transition pores 
 are impaired by dopamine oxidation products   making it plausible that the early damage of mitochondria observed in this 
 cellular model reflects the actions of -synuclein protofibrils and the subsequent increase of the membrane 
 permeability in the presence of oxidized catecholamine metabolites . we further analyzed the resting membrane potential and whole - cell 
 membrane conductance using the ramp voltage in the cell lines 
 expressing wild - type or mutant -synuclein . interestingly , the cells expressing a53 t -synuclein have the most depolarized membrane potential . by the application of the 
 ramp voltage under the whole cell voltage - clamp condition , we 
 obtained an almost linear current - voltage ( i - v ) relationship in 
 each cell line . the slope of the i - v relationship in the cells 
 expressing mutant -synuclein was significantly steeper than that in the cells expressing the vector alone or wild - type -synuclein , indicating that the 
 expression of mutant -synuclein results in higher ion permeability of the plasma membrane . because it 
 has been suggested that abnormal intracellular calcium homeostasis 
 plays a crucial role in the pathogenesis of neurodegenerative 
 disorders , the intracellular free calcium concentrations in -synuclein - transfected cells were quantified using a 
 calcium indicator dye , fura-2 . notably , both the intracellular calcium concentrations under basal conditions and 
 after depolarization induced by potassium chloride application 
 were significantly higher in the mutant -synuclein expressing cells than in cells expressing the empty vector or 
 wild - type -synuclein . these results suggest that mutant -synuclein is involved in the perturbation of the intracellular calcium homeostasis . taken together , our data from cellular models of synucleinopathy 
 suggest that oligomer or protofibril , but not aggregate or fibril , 
 formation of -synuclein plays a key role in the 
 pathomechanisms of synucleinopathy ( figure 1 ) . the 
 iron specifically triggers the aggregate formation of 
 -synuclein , but this seems to be a cytoprotective process 
 . the cytotoxic protofibril formation may be facilitated by not only gene mutations , but also the modification of 
 -synuclein by catechol - derived quinones [ 28 , 32 ] . the cellular membranes are the primary targets injured by protofibrils 
 , and the mitochondrial dysfunction seems to be an initial step in the neurodegeneration of synucleinopathy 
 . obviously , protofibrils or oligomers of -synuclein are heterogeneous in size and stability and 
 exist as mixtures in the cytosol . therefore , at present it is 
 difficult to specify the detailed molecular structures that may be 
 responsible for the cellular injuries . however , from these data , 
 it is plausible that the reduction of the protofibril pool may 
 rescue neurons from death ( figure 1 ) . if this is the 
 case , not only the acceleration of degradation ( c in figure 1 ) but also the facilitation of aggregate 
 formation ( d in figure 1 ) , may be a novel strategy for the treatment of synucleinopathy . of course , the most important 
 way would be to decrease the input ( a in figure 1 ) 
 into the protofibril pool . the inclusions in synucleinopathies were proved to be composed of -sheet rich fibrils formed by nitrated species of 
 -synuclein . several lines of evidence suggested that reactive oxygen species ( ros ) play a key role in the 
 conformational change of -synuclein and the following 
 aggregate formation [ 1215 ] . we developed human dopaminergic sh - sy5y cells overexpressing wild - type or mutant -synucleins , and established experimental models of 
 intracellular aggregate formation following the exposure to 
 various ros . the aggregates thus formed were 
 immunopositive for ubiquitin , nitrotyrosine , and dityrosine , and positive for thioflavin s staining , which was in good agreement with the pathological features of 
 inclusion bodies in synucleinopathies . the 
 -tubulin and molecular chaperones coexisted as well , 
 suggesting that the aggregate formation was associated with the 
 intracellular transport system for protein turnover responses 
 against the toxic effects of misfolded proteins . such mechanisms 
 are called  aggresome  and are suggested to represent one of the 
 cytoprotective responses [ 1618 ] . interestingly , the recent study on huntingtin showed that inclusion body formation 
 reduced the risk of neuronal death . however , it is still controversial whether the aggregate formation of 
 -synuclein has cytotoxicity in the neuronal cell or 
 sequesters toxic species . we established a cellular model in which intracellular 
 -synuclein aggregations were efficiently formed in 
 response to various types of ros exposure [ 9 , 19 ] . under these 
 conditions , a significant number of cells showed caspase 3 
 activation . to explore possible relationships between the aggregate formation and   apoptosis , first we investigated 
 whether -synuclein aggregates colocalized with activated 
 caspase-3 using a double immunostaining method . following the 
 combined exposure of the cells to a no donor and rotenone , 
 -synuclein aggregates were efficiently formed in the 
 cytoplasm as previously reported . surprisingly , 
 immunocytochemical analyses revealed that the aggregate positive 
 cells did not show any caspase 3 activations and , conversely , that 
 caspase 3 activated cells did not contain any -synuclein aggregates . iron was able to induce -synuclein aggregates more effectively than any other ros inducers 
 and no donors , suggesting the iron plays a key role in the 
 aggregate formation . when using both ros and no inducers , the addition of ferric iron triggered further aggregate formation , 
 but cells positive for activated caspase 3 were not coincident 
 with aggregate positive cells . in quantification experiments , it 
 was revealed that caspase 3-positive cells were decreased by the 
 addition of ferric iron . on the other hand , by chelating ferric 
 iron , the aggregate formation was decreased with concomitant 
 increases of caspase 3 activation . these data suggest that the 
 ferric iron plays a key role in the -synuclein aggregation . furthermore , these data also imply that the aggregate 
 formation may be cytoprotective against various cellular insults 
 including oxidative stress [ 19 , 20 ] . since -synuclein is ubiquitously expressed at 
 high levels in all brain regions , the mechanisms responsible for the preferential and selective neurodegeneration 
 of dopaminergic neurons in the substantia nigra remain to be 
 determined . previous studies suggested that the specific 
 vulnerability of dopaminergic neurons may be linked to the 
 cytotoxic oxidative potential of dopamine . highly reactive oxygen species ( ros ) are generated not only in dopamine 
 oxidation but also during the decay of catechol - derived 
 orthoquinones which covalently incorporate into a variety of 
 molecules including proteins and nucleic acids . on the other hand , previous reports demonstrated that -synuclein 
 might regulate dopamine metabolism by direct interaction with the 
 tyrosine hydroxylase , the dopamine transporter 
 and vesicular monoamine transporter ( vmat2 ) , key proteins in the 
 regulation of the dopamine content within nerve terminals 
 [ 26 , 27 ] . therefore , the pathological metabolism of -synuclein may be closely linked to the misregulation of 
 dopamine , consequently leading to neuronal death . in support of 
 this notion , catechol - derived orthoquinones ( eg , dopamine - quinone 
 or dopa - quinone ) accelerate and stabilize the formation of 
 -synuclein protofibrils by inhibiting the conversion of 
 toxic protofibrils into fibrils [ 28 , 29 ] . to shed light on the pathophysiological mechanisms underlying 
 -synuclein - mediated neurodegeneration in dopamine neurons , 
 we developed novel neuronal cell lines coexpressing 
 -synuclein ( wild - type or a53 t ) and tyrosinase that 
 produces catecholamines and their oxidized metabolites [ 30 , 31 ] . investigating the effects of wild - type or mutant -synuclein expression , we found that the coexpression of 
 wild - type and a53 t mutant -synuclein in tyrosinase - overexpressing cells exacerbated dna damage and 
 successive apoptotic cell death compared to the cells 
 overexpressing cat or antisense -synuclein . both wild - type 
 and a53 t mutant -synucleins coexpressed with tyrosinase 
 resulted in the gradual accumulation of high - molecular weight 
 complexes immunopositive for -synuclein . this band , 
 possibly representing oligomerized forms , corresponded to the size 
 of -synuclein tetramer and was also detected by the 
 nbt / glycinate redox - cycling staining , suggesting that it was 
 modified by quinones . moreover , during these processes , the mitochondrial membrane 
 potential was specifically decreased without the activation of map 
 kinases . although the underlying mechanism(s ) 
 of neuronal cell death following the coexpression of tyrosinase and 
 -synuclein are still elusive , it is likely that 
 -synuclein modified by the oxidized catechol metabolites 
 forms cytotoxic intermediates , that is ,  protofibrils  . recent 
 reports suggested that protofibrillar -synuclein tightly binds to lipid bilayers and 
 increases the membrane permeability by forming pore - like structures 
 [ 3335 ] . while the membranous structures damaged by protofibrils in dopaminergic nerve 
 terminals remain unknown , intracellular organelles , such as synaptic vesicles and mitochondria , 
 are possible candidates . in this regard , disruption of synaptic 
 vesicle membranes would result in an increase of the cytoplasmic 
 dopamine levels that would trigger the further accumulation of 
 dopamine - quinone and dopamine - derived oxyradicals and thus lead to 
 a vicious cycle . likewise , mitochondrial enzymes in the electron 
 transport chain and the functional permeability transition pores 
 are impaired by dopamine oxidation products   making it plausible that the early damage of mitochondria observed in this 
 cellular model reflects the actions of -synuclein protofibrils and the subsequent increase of the membrane 
 permeability in the presence of oxidized catecholamine metabolites . we further analyzed the resting membrane potential and whole - cell 
 membrane conductance using the ramp voltage in the cell lines 
 expressing wild - type or mutant -synuclein . interestingly , the cells expressing a53 t -synuclein have the most depolarized membrane potential . by the application of the 
 ramp voltage under the whole cell voltage - clamp condition , we 
 obtained an almost linear current - voltage ( i - v ) relationship in 
 each cell line . the slope of the i - v relationship in the cells 
 expressing mutant -synuclein was significantly steeper than that in the cells expressing the vector alone or wild - type -synuclein , indicating that the 
 expression of mutant -synuclein results in higher ion permeability of the plasma membrane . because it 
 has been suggested that abnormal intracellular calcium homeostasis 
 plays a crucial role in the pathogenesis of neurodegenerative 
 disorders , the intracellular free calcium concentrations in -synuclein - transfected cells were quantified using a 
 calcium indicator dye , fura-2 . notably , both the intracellular calcium concentrations under basal conditions and 
 after depolarization induced by potassium chloride application 
 were significantly higher in the mutant -synuclein expressing cells than in cells expressing the empty vector or 
 wild - type -synuclein . these results suggest that mutant -synuclein is involved in the perturbation of the intracellular calcium homeostasis . taken together , our data from cellular models of synucleinopathy 
 suggest that oligomer or protofibril , but not aggregate or fibril , 
 formation of -synuclein plays a key role in the 
 pathomechanisms of synucleinopathy ( figure 1 ) . the 
 iron specifically triggers the aggregate formation of 
 -synuclein , but this seems to be a cytoprotective process 
 . the cytotoxic protofibril formation may be facilitated by not only gene mutations , but also the modification of 
 -synuclein by catechol - derived quinones [ 28 , 32 ] . the cellular membranes are the primary targets injured by protofibrils 
 , and the mitochondrial dysfunction seems to be an initial step in the neurodegeneration of synucleinopathy 
 . obviously , protofibrils or oligomers of -synuclein are heterogeneous in size and stability and 
 exist as mixtures in the cytosol . therefore , at present it is 
 difficult to specify the detailed molecular structures that may be 
 responsible for the cellular injuries . however , from these data , 
 it is plausible that the reduction of the protofibril pool may 
 rescue neurons from death ( figure 1 ) . if this is the 
 case , not only the acceleration of degradation ( c in figure 1 ) but also the facilitation of aggregate 
 formation ( d in figure 1 ) , may be a novel strategy for the treatment of synucleinopathy . of course , the most important 
 way would be to decrease the input ( a in figure 1 ) 
 into the protofibril pool .
-synuclein is a key molecule in the pathogenesis of 
 synucleinopathy including parkinson 's disease and multiple system 
 atrophy . in this mini - review 
 , we mainly focus on recent data 
 obtained from cellular models of synucleinopathy and discuss the 
 possible mechanisms of neurodegeneration . 
 recent progress suggests 
 that the aggregate formation of -synuclein is cytoprotective and that its precursor oligomer ( protofibril ) may 
 be cytotoxic . 
 the catechol - derived quinones are the candidate 
 molecules that facilitate the oligomer formation of -synuclein . 
 furthermore , the cellular membranes are shown to be 
 the primary targets injured by mutant -synucleins , and 
 the mitochondrial dysfunction seems to be an initial step in the neuronal death .