parkinson s disease ( pd ) is the second most frequent age - associated brain degeneration disorder , affecting about 1  % of the population over 65  years of age .
the pd - specific progressive movement deficit is mostly due to the severe affliction and cell death of midbrain nigrostriatal dopaminergic neurons .
surviving neurons in vulnerable regions exhibit aggregates predominantly consisting of the protein alpha - synuclein , which are visualized as lewy neurites and lewy bodies , both in sporadic late - onset and most familial early onset pd variants .
autosomal dominant pd with early clinical manifestation was observed in rare families , leading to the identification of alpha - synuclein ( snca ) protein missense mutations such as a53 t ( termed the park1 variant ) and of snca gene duplication / triplication events ( park4 variant ) as the strongest causes of this pathology [ 3 , 4 ] .
further recruitment of parkinson s families enabled identification of a list of disease genes responsible for monogenic pd .
in addition , recent characterization of very large collectives of late - manifesting sporadic pd cases through genome - wide allele association studies ( gwas ) identified two regions on chromosome 4 ( snca and gak / cplx1 loci ) that contain genetic variants predisposing to multifactorial pd .
variations in the snca gene 3-untranslated region ( 3-utr ) and its promoter correlated strongly with pd risk .
it is associated with the lipid membranes of synaptic vesicles and interacts with synaptobrevin , a component of the snare complex , mediating vesicle exocytosis and neurotransmitter release .
its toxic gain - of - function leads over time to impaired synaptic vesicle release and synaptic failure [ 9 , 10 ] .
current investigations aim to elucidate alpha - synuclein - triggered pathology , concentrating on disease stages before the occurrence of irreversible cell loss , when neuroprotective therapies might still be efficacious . here , we focused on two independent mouse lines of inbred fvb / n background with ~1.5-fold overexpression of human a53t - alpha - synuclein in nigrostriatal dopaminergic neurons under control of the heterologous neuron - specific prion - promoter .
a53t - alpha - synuclein overexpression in these mice occurs in presynaptic nigral dopaminergic neurons and presynaptic cortical glutamatergic neurons , but not in postsynaptic striatal neurons .
these mice display apparently normal movements at age 6  months , but progress to significantly impaired spontaneous locomotion by age 18  months , despite the absence of neuronal loss in the nigrostriatal projection .
previous expression profiling in these mice identified a homer-1a transcript dysregulation throughout the brain and a 14 - 3 - 3 epsilon protein upregulation selectively in the striatum as molecular effects of alpha - synuclein triggered pathology .
the alterations in these signalling molecules were temporally correlated with reduced striatal dopamine release and deficient long - term depression [ 1214 , 9 ] .
to gain insight into the mechanisms underlying the impairment in vesicle exocytosis and neurotransmitter release , we surveyed progressive expression changes in midbrain / brainstem tissue using genome - wide unbiased transcriptome profiling .
previously documented ( geo database gse20547 , see also ) global transcriptome data from striatum , midbrain / brainstem and cerebellum of human a53t - alpha - synuclein overexpressing mice were filtered .
we selected those significant changes at age 18  months relative to age 6  months , which were midbrain / brainstem - specific and were consistent between both transgenic mouse lines ( prpmta and prpmtb ) .
further selection prioritized those transcripts with no corresponding significant changes in wild - type midbrain / brainstem and in wild - type / transgenic striatum and cerebellum , resulting in the identification of 49 candidate effects of synucleinopathy ( table  1 ) .
among the progressive upregulation effects , the increase of foxp1 mrna levels by a53t - alpha - synuclein overexpression was particularly interesting in view of our previous finding that foxp1 ( encoding forkhead box p1 ) is downregulated in alpha - synuclein knockout mouse .
thus , the midbrain - identity - mediating transcription factor foxp1 appears to depend in its brain levels both on the gain - of - function and the loss - of - function of alpha - synuclein . among the progressive downregulation effects , the decreased levels of cplx1 ( encoding complexin-1 )
selectively in the mutant midbrain / brainstem were especially interesting , in view of the co - localization of alpha - synuclein and complexin-1 at the snare complex .
other midbrain / brainstem - selective significant dysregulations of vesicle endocytosis / exocytosis pathway factors included the downregulation of rabl2a transcript and the upregulation of rabgef1 , in good agreement with previous observations that alpha - synuclein disrupts cellular rab homeostasis .
more ubiquitous dysregulations were detectable for three 14 - 3 - 3 isoforms ( table  1 bottom ) , which are established downstream targets of alpha - synuclein function .
14 - 3 - 3 family isoforms heterodimerize to protect phosphoserine - phosphothreonine groups and are ( 1 ) sequence homologous to alpha - synuclein , ( 2 ) putative direct interactor proteins of alpha - synuclein , ( 3 ) expression - modulated by alpha - synuclein , ( 4 ) sequestered into the lewy bodies , ( 5 ) able to change their levels in response to pathogenic alpha - synuclein mutations and thereby modulate neurotoxicity [ 1720 ] .
thus , at least seven of the expression changes are credible in the light of their function and of previous knowledge about alpha - synuclein .
all information per animal including age and sex with the resulting microarray data discussed in this publication is deposited in the ncbi 's database gene expression omnibus ( geo ) and is accessible through geo series accession number gse20547 .
validation studies of cplx1 mrna expression in independent tissues by qpcr confirmed the significant downregulation in midbrain / brainstem of prpmta mice ( fig . 
1).table 1global transcriptome analysis of mice with nigrostriatal overexpression of human a53t - alpha - synuclein showing significant changes from age 6 to 18 +  monthsthe upper rows show all 49 genes with known functions , which exhibited significant and consistent progression changes in both transgenic midbrain / brainstem tissues , but not in wild - type midbrain / brainstem or striatum or cerebellum .
grey background with bold gene symbol and comments were used to highlight the most promising novel expression effect of synucleinopathy , cplx1 ( encoding complexin-1 ) .
the lower rows show known expression effects of synucleinopathy for comparison , highlighting the best previously established transcript ywhae ( encoding 14 - 3 - 3epsilon ) .
column ( a ) documents the affymetrix probeset i d ; ( b , c ) the relative expression ( re ) values for transgenic lines prpmta and prpmtb , respectively , highlighting changes > 1.7 or < 0.6 in bold letters ; ( d ) the adjusted p value to judge significance after correction for multiple testing ; ( e  g ) the lack of significant changes ( 0 ) in striatum ( s ) of wild type ( wt ) and the two transgenic lines ( a and b ) , respectively ; ( h  j ) the lack of significant changes in wt compared to significant upregulations ( 1 ) or downregulations ( 1 ) in midbrain / brainstem ( mb ) tissue of two transgenic lines a and b , respectively ; ( k  m ) the lack of significant changes in cerebellum ( c ) of wild type and two transgenic lines a and b , respectively ; ( n ) the gene symbol to access genecards and ncbi online databases using different background colours to emphasize functional pathways in common between individual genes ; ( o ) authors summaries on the functions of each gene product with respect to synaptic failure , according to genecards and pubmed online databases .
the rows of the upper table part were ordered from top in descending significance 
 fig .
1quantitative real - time reverse transcriptase pcr demonstrates reduced mrna levels of complexin-1 in the midbrain / brainstem of mice with a53t - alpha - synuclein overexpression .
tbp was always used as loading control , and mrna level ratios were normalized to wt .
complexin-1 transcript was specifically detected by a custom - made taqman assay , using midbrain / brainstem extracts from the transgenic line prpmta versus wild type ( wt ) ( n  =  18 versus 15 ) at age 18  months , demonstrating a significant downregulation in prpmta tissue 
  global transcriptome analysis of mice with nigrostriatal overexpression of human a53t - alpha - synuclein showing significant changes from age 6 to 18 +  months the upper rows show all 49 genes with known functions , which exhibited significant and consistent progression changes in both transgenic midbrain / brainstem tissues , but not in wild - type midbrain / brainstem or striatum or cerebellum .
grey background with bold gene symbol and comments were used to highlight the most promising novel expression effect of synucleinopathy , cplx1 ( encoding complexin-1 ) .
the lower rows show known expression effects of synucleinopathy for comparison , highlighting the best previously established transcript ywhae ( encoding 14 - 3 - 3epsilon ) .
column ( a ) documents the affymetrix probeset i d ; ( b , c ) the relative expression ( re ) values for transgenic lines prpmta and prpmtb , respectively , highlighting changes > 1.7 or < 0.6 in bold letters ; ( d ) the adjusted p value to judge significance after correction for multiple testing ; ( e  g ) the lack of significant changes ( 0 ) in striatum ( s ) of wild type ( wt ) and the two transgenic lines ( a and b ) , respectively ; ( h  j ) the lack of significant changes in wt compared to significant upregulations ( 1 ) or downregulations ( 1 ) in midbrain / brainstem ( mb ) tissue of two transgenic lines a and b , respectively ; ( k  m ) the lack of significant changes in cerebellum ( c ) of wild type and two transgenic lines a and b , respectively ; ( n ) the gene symbol to access genecards and ncbi online databases using different background colours to emphasize functional pathways in common between individual genes ; ( o ) authors summaries on the functions of each gene product with respect to synaptic failure , according to genecards and pubmed online databases .
the rows of the upper table part were ordered from top in descending significance quantitative real - time reverse transcriptase pcr demonstrates reduced mrna levels of complexin-1 in the midbrain / brainstem of mice with a53t - alpha - synuclein overexpression .
tbp was always used as loading control , and mrna level ratios were normalized to wt .
complexin-1 transcript was specifically detected by a custom - made taqman assay , using midbrain / brainstem extracts from the transgenic line prpmta versus wild type ( wt ) ( n  =  18 versus 15 ) at age 18  months , demonstrating a significant downregulation in prpmta tissue we focused on the downregulation of midbrain / brainstem cplx1 mrna as a novel and promising effect , since the encoded protein complexin-1 is involved in the stimulus - dependent control of secretory vesicle exocytosis through the snare complex [ 21 , 22 ] .
alpha - synuclein was also shown to modulate snare assembly and vesicle clustering , so this expression effect might constitute a very direct and early consequence of alpha - synuclein mutations .
densitometric analysis of immunoblots revealed a significant increase of complexin-1 protein levels in the midbrain / brainstem of aged a53t - alpha - synuclein overexpressing mice ( fig . 
the alterations were readily apparent by ecl detection of membranes , making more sophisticated approaches such as near - infrared immunoblot detection or quantification by elisa unnecessary.fig .
2quantitative immunoblots demonstrate dysregulated levels of complexin-1 and 14 - 3 - 3epsilon proteins in the midbrain / brainstem of mice with alpha - synuclein mutation .
beta - actin was always used as loading control , and protein level ratios were normalized to wt .
representative membranes are shown at the left , bar graph statistics of quantification at the right .
* p value < 0.05 , * * p  <  0.01 and * * * p  <  0.001 . a complexin-1 and complexin-2 were detected with the antibody from sysy , using midbrain / brainstem protein extracts from the transgenic line prpmta versus wild type ( wt ) ( n  =  3 versus 4 ) at age 18  months , demonstrating significantly increased complexin-1 levels .
b midbrain / brainstem protein from transgenic line prpmtb versus wild type ( wt ) ( four vs. three ) at age 18  months also showed significantly increased complexin-1 levels .
c in comparison , selective detection of 14 - 3 - 3epsilon abundance change ( five vs. five ) as a repeatedly published molecular effect of alpha - synucleinopathy failed to reveal changes in protein levels , in spite of its significantly changed mrna levels in mouse midbrain / brainstem ( table  1 ) .
d levels of complexin-1 and complexin-2 ( antibody from sysy ) were significantly increased in alpha - synuclein knockout mice ( snca ko ) at age 3  months ( five ko vs. four wt ) , in inverse correlation to alpha - synuclein levels , demonstrating that complexin levels respond not only to the toxic alpha - synuclein gain - of - function / aggregation process but also to its loss - of - function .
e significant downregulation of 14 - 3 - 3epsilon ( five ko vs. four wt ) .
these data indicate that 14 - 3 - 3epsilon protein levels are directly correlated to the loss - of - function of alpha - synuclein 
  quantitative immunoblots demonstrate dysregulated levels of complexin-1 and 14 - 3 - 3epsilon proteins in the midbrain / brainstem of mice with alpha - synuclein mutation .
beta - actin was always used as loading control , and protein level ratios were normalized to wt .
representative membranes are shown at the left , bar graph statistics of quantification at the right .
* p value < 0.05 , * * p  <  0.01 and * * * p  <  0.001 . a complexin-1 and complexin-2 were detected with the antibody from sysy , using midbrain / brainstem protein extracts from the transgenic line prpmta versus wild type ( wt ) ( n  =  3 versus 4 ) at age 18  months , demonstrating significantly increased complexin-1 levels .
b midbrain / brainstem protein from transgenic line prpmtb versus wild type ( wt ) ( four vs. three ) at age 18  months also showed significantly increased complexin-1 levels .
c in comparison , selective detection of 14 - 3 - 3epsilon abundance change ( five vs. five ) as a repeatedly published molecular effect of alpha - synucleinopathy failed to reveal changes in protein levels , in spite of its significantly changed mrna levels in mouse midbrain / brainstem ( table  1 ) .
d levels of complexin-1 and complexin-2 ( antibody from sysy ) were significantly increased in alpha - synuclein knockout mice ( snca ko ) at age 3  months ( five ko vs. four wt ) , in inverse correlation to alpha - synuclein levels , demonstrating that complexin levels respond not only to the toxic alpha - synuclein gain - of - function / aggregation process but also to its loss - of - function .
e significant downregulation of 14 - 3 - 3epsilon ( five ko vs. four wt ) .
these data indicate that 14 - 3 - 3epsilon protein levels are directly correlated to the loss - of - function of alpha - synuclein these data obtained in a53t - alpha - synuclein overexpressing midbrain / brainstem complement previous observations from alpha-/beta - synuclein double - null mice , which exhibit upregulated complexin-1 and downregulated 14 - 3 - 3 epsilon protein in the whole brain . to test whether alpha - synuclein or beta - synuclein is responsible for the observed changes , we studied midbrain / brainstem from mice with snca knockout in 129/svev background   and demonstrated significant upregulation for complexin-1 and downregulation for 14 - 3 - 3epsilon protein ( fig . 
thus , 14 - 3 - 3 isoform and complexin-1 protein levels respond not only to toxic gain - of - function mutations in alpha - synuclein but also to its loss - of - physiological function .
previously documented ( geo database gse20547 , see also ) global transcriptome data from striatum , midbrain / brainstem and cerebellum of human a53t - alpha - synuclein overexpressing mice were filtered .
we selected those significant changes at age 18  months relative to age 6  months , which were midbrain / brainstem - specific and were consistent between both transgenic mouse lines ( prpmta and prpmtb ) .
further selection prioritized those transcripts with no corresponding significant changes in wild - type midbrain / brainstem and in wild - type / transgenic striatum and cerebellum , resulting in the identification of 49 candidate effects of synucleinopathy ( table  1 ) .
among the progressive upregulation effects , the increase of foxp1 mrna levels by a53t - alpha - synuclein overexpression was particularly interesting in view of our previous finding that foxp1 ( encoding forkhead box p1 ) is downregulated in alpha - synuclein knockout mouse .
thus , the midbrain - identity - mediating transcription factor foxp1 appears to depend in its brain levels both on the gain - of - function and the loss - of - function of alpha - synuclein . among the progressive downregulation effects , the decreased levels of cplx1 ( encoding complexin-1 )
selectively in the mutant midbrain / brainstem were especially interesting , in view of the co - localization of alpha - synuclein and complexin-1 at the snare complex .
other midbrain / brainstem - selective significant dysregulations of vesicle endocytosis / exocytosis pathway factors included the downregulation of rabl2a transcript and the upregulation of rabgef1 , in good agreement with previous observations that alpha - synuclein disrupts cellular rab homeostasis .
more ubiquitous dysregulations were detectable for three 14 - 3 - 3 isoforms ( table  1 bottom ) , which are established downstream targets of alpha - synuclein function .
14 - 3 - 3 family isoforms heterodimerize to protect phosphoserine - phosphothreonine groups and are ( 1 ) sequence homologous to alpha - synuclein , ( 2 ) putative direct interactor proteins of alpha - synuclein , ( 3 ) expression - modulated by alpha - synuclein , ( 4 ) sequestered into the lewy bodies , ( 5 ) able to change their levels in response to pathogenic alpha - synuclein mutations and thereby modulate neurotoxicity [ 1720 ] .
thus , at least seven of the expression changes are credible in the light of their function and of previous knowledge about alpha - synuclein .
all information per animal including age and sex with the resulting microarray data discussed in this publication is deposited in the ncbi 's database gene expression omnibus ( geo ) and is accessible through geo series accession number gse20547 .
validation studies of cplx1 mrna expression in independent tissues by qpcr confirmed the significant downregulation in midbrain / brainstem of prpmta mice ( fig . 
1).table 1global transcriptome analysis of mice with nigrostriatal overexpression of human a53t - alpha - synuclein showing significant changes from age 6 to 18 +  monthsthe upper rows show all 49 genes with known functions , which exhibited significant and consistent progression changes in both transgenic midbrain / brainstem tissues , but not in wild - type midbrain / brainstem or striatum or cerebellum .
grey background with bold gene symbol and comments were used to highlight the most promising novel expression effect of synucleinopathy , cplx1 ( encoding complexin-1 ) .
the lower rows show known expression effects of synucleinopathy for comparison , highlighting the best previously established transcript ywhae ( encoding 14 - 3 - 3epsilon ) .
column ( a ) documents the affymetrix probeset i d ; ( b , c ) the relative expression ( re ) values for transgenic lines prpmta and prpmtb , respectively , highlighting changes > 1.7 or < 0.6 in bold letters ; ( d ) the adjusted p value to judge significance after correction for multiple testing ; ( e  g ) the lack of significant changes ( 0 ) in striatum ( s ) of wild type ( wt ) and the two transgenic lines ( a and b ) , respectively ; ( h  j ) the lack of significant changes in wt compared to significant upregulations ( 1 ) or downregulations ( 1 ) in midbrain / brainstem ( mb ) tissue of two transgenic lines a and b , respectively ; ( k  m ) the lack of significant changes in cerebellum ( c ) of wild type and two transgenic lines a and b , respectively ; ( n ) the gene symbol to access genecards and ncbi online databases using different background colours to emphasize functional pathways in common between individual genes ; ( o ) authors summaries on the functions of each gene product with respect to synaptic failure , according to genecards and pubmed online databases .
the rows of the upper table part were ordered from top in descending significance 
 fig .
1quantitative real - time reverse transcriptase pcr demonstrates reduced mrna levels of complexin-1 in the midbrain / brainstem of mice with a53t - alpha - synuclein overexpression .
tbp was always used as loading control , and mrna level ratios were normalized to wt .
complexin-1 transcript was specifically detected by a custom - made taqman assay , using midbrain / brainstem extracts from the transgenic line prpmta versus wild type ( wt ) ( n  =  18 versus 15 ) at age 18  months , demonstrating a significant downregulation in prpmta tissue 
  global transcriptome analysis of mice with nigrostriatal overexpression of human a53t - alpha - synuclein showing significant changes from age 6 to 18 +  months the upper rows show all 49 genes with known functions , which exhibited significant and consistent progression changes in both transgenic midbrain / brainstem tissues , but not in wild - type midbrain / brainstem or striatum or cerebellum .
grey background with bold gene symbol and comments were used to highlight the most promising novel expression effect of synucleinopathy , cplx1 ( encoding complexin-1 ) .
the lower rows show known expression effects of synucleinopathy for comparison , highlighting the best previously established transcript ywhae ( encoding 14 - 3 - 3epsilon ) .
column ( a ) documents the affymetrix probeset i d ; ( b , c ) the relative expression ( re ) values for transgenic lines prpmta and prpmtb , respectively , highlighting changes > 1.7 or < 0.6 in bold letters ; ( d ) the adjusted p value to judge significance after correction for multiple testing ; ( e  g ) the lack of significant changes ( 0 ) in striatum ( s ) of wild type ( wt ) and the two transgenic lines ( a and b ) , respectively ; ( h  j ) the lack of significant changes in wt compared to significant upregulations ( 1 ) or downregulations ( 1 ) in midbrain / brainstem ( mb ) tissue of two transgenic lines a and b , respectively ; ( k  m ) the lack of significant changes in cerebellum ( c ) of wild type and two transgenic lines a and b , respectively ; ( n ) the gene symbol to access genecards and ncbi online databases using different background colours to emphasize functional pathways in common between individual genes ; ( o ) authors summaries on the functions of each gene product with respect to synaptic failure , according to genecards and pubmed online databases .
the rows of the upper table part were ordered from top in descending significance quantitative real - time reverse transcriptase pcr demonstrates reduced mrna levels of complexin-1 in the midbrain / brainstem of mice with a53t - alpha - synuclein overexpression .
tbp was always used as loading control , and mrna level ratios were normalized to wt .
complexin-1 transcript was specifically detected by a custom - made taqman assay , using midbrain / brainstem extracts from the transgenic line prpmta versus wild type ( wt ) ( n  =  18 versus 15 ) at age 18  months , demonstrating a significant downregulation in prpmta tissue
we focused on the downregulation of midbrain / brainstem cplx1 mrna as a novel and promising effect , since the encoded protein complexin-1 is involved in the stimulus - dependent control of secretory vesicle exocytosis through the snare complex [ 21 , 22 ] .
alpha - synuclein was also shown to modulate snare assembly and vesicle clustering , so this expression effect might constitute a very direct and early consequence of alpha - synuclein mutations .
densitometric analysis of immunoblots revealed a significant increase of complexin-1 protein levels in the midbrain / brainstem of aged a53t - alpha - synuclein overexpressing mice ( fig . 
the alterations were readily apparent by ecl detection of membranes , making more sophisticated approaches such as near - infrared immunoblot detection or quantification by elisa unnecessary.fig .
2quantitative immunoblots demonstrate dysregulated levels of complexin-1 and 14 - 3 - 3epsilon proteins in the midbrain / brainstem of mice with alpha - synuclein mutation .
beta - actin was always used as loading control , and protein level ratios were normalized to wt .
representative membranes are shown at the left , bar graph statistics of quantification at the right .
* p value < 0.05 , * * p  <  0.01 and * * * p  <  0.001 . a complexin-1 and complexin-2 were detected with the antibody from sysy , using midbrain / brainstem protein extracts from the transgenic line prpmta versus wild type ( wt ) ( n  =  3 versus 4 ) at age 18  months , demonstrating significantly increased complexin-1 levels .
b midbrain / brainstem protein from transgenic line prpmtb versus wild type ( wt ) ( four vs. three ) at age 18  months also showed significantly increased complexin-1 levels .
c in comparison , selective detection of 14 - 3 - 3epsilon abundance change ( five vs. five ) as a repeatedly published molecular effect of alpha - synucleinopathy failed to reveal changes in protein levels , in spite of its significantly changed mrna levels in mouse midbrain / brainstem ( table  1 ) .
d levels of complexin-1 and complexin-2 ( antibody from sysy ) were significantly increased in alpha - synuclein knockout mice ( snca ko ) at age 3  months ( five ko vs. four wt ) , in inverse correlation to alpha - synuclein levels , demonstrating that complexin levels respond not only to the toxic alpha - synuclein gain - of - function / aggregation process but also to its loss - of - function .
e significant downregulation of 14 - 3 - 3epsilon ( five ko vs. four wt ) .
these data indicate that 14 - 3 - 3epsilon protein levels are directly correlated to the loss - of - function of alpha - synuclein 
  quantitative immunoblots demonstrate dysregulated levels of complexin-1 and 14 - 3 - 3epsilon proteins in the midbrain / brainstem of mice with alpha - synuclein mutation .
beta - actin was always used as loading control , and protein level ratios were normalized to wt .
representative membranes are shown at the left , bar graph statistics of quantification at the right . * p value < 0.05 , * * p  <  0.01 and * * * p  <  0.001 .
a complexin-1 and complexin-2 were detected with the antibody from sysy , using midbrain / brainstem protein extracts from the transgenic line prpmta versus wild type ( wt ) ( n  =  3 versus 4 ) at age 18  months , demonstrating significantly increased complexin-1 levels . b midbrain / brainstem protein from transgenic line prpmtb versus wild type ( wt ) ( four vs. three ) at age 18  months
c in comparison , selective detection of 14 - 3 - 3epsilon abundance change ( five vs. five ) as a repeatedly published molecular effect of alpha - synucleinopathy failed to reveal changes in protein levels , in spite of its significantly changed mrna levels in mouse midbrain / brainstem ( table  1 ) .
d levels of complexin-1 and complexin-2 ( antibody from sysy ) were significantly increased in alpha - synuclein knockout mice ( snca ko ) at age 3  months ( five ko vs. four wt ) , in inverse correlation to alpha - synuclein levels , demonstrating that complexin levels respond not only to the toxic alpha - synuclein gain - of - function / aggregation process but also to its loss - of - function .
e significant downregulation of 14 - 3 - 3epsilon ( five ko vs. four wt ) .
these data indicate that 14 - 3 - 3epsilon protein levels are directly correlated to the loss - of - function of alpha - synuclein
these data obtained in a53t - alpha - synuclein overexpressing midbrain / brainstem complement previous observations from alpha-/beta - synuclein double - null mice , which exhibit upregulated complexin-1 and downregulated 14 - 3 - 3 epsilon protein in the whole brain . to test whether alpha - synuclein or beta - synuclein is responsible for the observed changes , we studied midbrain / brainstem from mice with snca knockout in 129/svev background   and demonstrated significant upregulation for complexin-1 and downregulation for 14 - 3 - 3epsilon protein ( fig . 
isoform and complexin-1 protein levels respond not only to toxic gain - of - function mutations in alpha - synuclein but also to its loss - of - physiological function .
our data confirm previous findings that alpha - synuclein abundance modulates the levels of 14 - 3 - 3 isoforms .
it was previously known that cplx1 levels are altered in alpha + beta - synuclein double - knockout mice and that foxp1 mrna levels respond to the alpha - synuclein knockout .
we now report novel findings that also pathogenic gain - of - function mutations of alpha - synuclein have a modulatory role on cplx1 and foxp1 in mice that showed no demonstrable alpha - synuclein aggregation in midbrain / brainstem during their lifespan and that cplx1 levels change in the alpha - synuclein single knockout mouse brain .
this suggests that both cplx1 and foxp1 may be useful to monitor early stages of alpha - synuclein pathology .
in contrast , cplx1 shows a more ubiquitous expression pattern , similar to alpha - synuclein . although both cplx1 and snca were mainly studied regarding presynaptic vesicle dynamics , their expression in brain is not much higher than in blood platelets , where they have a role in stimulus - dependent secretory vesicle exocytosis to control thrombus formation .
thus , cplx1 might have potential as biomarker to monitor an alpha - synuclein gain - of - function in peripheral tissues like blood .
although our experiments were focused on modelling monogenic alpha - synucleinopathy variants of pd ( park4/1 ) , we are confident that complexin-1 plays a role in the genetically heterogeneous common idiopathic pd . our data from alpha - synucleinopathy mouse models are consistent with a proteome survey of midbrain from sporadic pd patients , which reported significantly elevated levels for complexin-1 and a trend towards elevated levels of 14 - 3 - 3 epsilon .
furthermore , the gak / cplx1 locus on chromosome 4 carries risk variants for sporadic pd in gwas studies .
a plausible explanation might predict that excess alpha - synuclein at the snare complex interacts with cplx1 and impairs its degradation .
this could occur as part of a sequestration process during the formation of alpha - synuclein oligomers and aggregates , reflecting incipient formation of inclusion bodies known as  lewy neurites  or
it has been observed that this aggregation process starts in the presynapses and sequesters local proteins such as synapsin .
overall , the transcriptomic profiling of our park1/park4 mouse model identified plausible molecular correlates of early nigrostriatal dopaminergic neurotransmission deficits previously observed in this mouse . in conclusion ,
the transcriptomic profiling of mouse midbrain / brainstem tissue with alpha - synuclein pathology has provided credible insights into early steps of pathogenesis , before the advent of neurodegeneration .
complexin-1 may be a better read - out of alpha - synucleinopathy than the previous gold standard 14 - 3 - 3 .
mice were housed in accordance with the german animal welfare act , the council directive of 24 november 1986 ( 86/609/ewg ) with annex ii , the ets123 ( european convention for the protection of vertebrate animals ) and the eu directive 2010/63/eu for animal experiments at the felasa - certified central animal facility ( zfe ) of the frankfurt university medical school . 

mouse breeding and characterization with brain dissection was carried out as described in the literature [ 28 , 29 , 24 ] .
all studies of mouse mutants were in comparison with age- and sex - matched wt controls from the same inbred background line , which were bred and aged in parallel , under controlled light cycle , periodic health - monitoring , and individually ventilated cage housing .
dissection of brain regions occurred rapidly after cervical dislocation , placing the brain in a sagittal view to perform a coronal section from the hypophysis stem towards the caudal end of the hippocampus .
olfactory brain regions , the cerebral cortex , septal and thalamic tissue were removed from the ventral tissue block to isolate the striatum . to obtain midbrain / brainstem from the caudal tissue block , the cortical , dorsal thalamic and tectal tissues were removed , yielding the substantia nigra continuous with ventral tegmental area , red nucleus , mammillary nuclei and brainstem . for the dissection of the cerebellum
all tissues were snap - frozen in liquid nitrogen and then stored at 80  c .
the individual transcript expression validation on a steponeplus equipment ( appliedbiosystems ) employed taqman assays ( appliedbiosystems ) mm00447333_m1 ( snca ) , mm01198853_m1 ( cplx1 ) and mm00446973_m1 ( tbp ) , with quantitative real - time reverse transcriptase polymerase chain reaction ( qpcr ) conditions as recommended for these assays . 

genome - wide transcriptomics of mouse brain regions was performed with affymetrix oligonucleotide microarrays as previously reported .
frozen tissues were homogenized on ice in a glass - teflon douncer in ripa buffer with 50  mm tris 
hcl ( ph 8) , 150  mm nacl , 1  % np-40 , 0.5  % na - deoxycholate , 0.1  % sds and protease inhibitor cocktail ( roche ) .
total lysates were briefly sonicated on ice , and cell debris was removed by centrifugation .
sds  page - separated proteins ( 20  g / lane ) were blotted onto a pvdf membrane ( bio - rad ) and probed .
the following primary antibodies for mouse alpha - synuclein ( 1:1,000 bd biosciences 610786 ) , complexin-1 ( 1:500 acris ap51050pu - n and 1:1,000 sysy 122002 ) , 14 - 3 - 3epsilon / eta / zeta / beta / gamma / theta ( 1:1,000 santacruz sc1020 and others from cellsignaling ) , beta - actin ( 1:1,000 sigma a5441 ) were used with their corresponding secondary antibodies ( ge healthcare uk limited lna931v / ag for ecl - anti - mouse - hp from sheep and lna934v / ag for ecl - anti - rabbit - hp from donkey ) for 1  h. the detection was made with supersignal west pico ( thermo scientific ) , with varying exposure times to avoid film sensitivity or saturation problems as well as non - linear effects .
the images were digitalized on a scanner ( epson ) and densitometry performed with the proprietary imagemaster total lab 2.00 software ( amershampharmacia ) or the public imagej software .
after normalization of candidate protein values versus beta - actin values from the identical membrane in excel , the changes were evaluated in graphpad statistics and plotting . 

statistical analyses presented in bar graphs were performed by unpaired student s t tests and plotted with the prism 3 software ( graphpad , la jolla , ca , usa ) .
 as the second most frequent neurodegenerative disorder of the aging population , parkinson s disease ( pd ) is characterized by progressive deficits in spontaneous movement , atrophy of dopaminergic midbrain neurons and aggregation of the protein alpha - synuclein ( snca ) . to elucidate molecular events before irreversible cell death , we studied synucleinopathy - induced expression changes in mouse brain and identified 49 midbrain / brainstem - specific transcriptional dysregulations . in particular complexin-1 
 ( cplx1 ) , rabl2a and 14 - 3 - 3epsilon ( ywhae ) downregulation , as well as upregulation of the midbrain - specific factor forkhead box p1 ( foxp1 ) and of rabgef1 , were interesting as early mrna level effects of alpha - synuclein triggered pathology . 
 the protein levels of complexin-1 were elevated in midbrain / brainstem tissue of mice with a53t - snca overexpression and of mice with snca - knockout . 
 the response of cplx1 and foxp1 levels to snca deficiency supports the notion that these factors are regulated by altered physiological function of alpha - synuclein . 
 thus , their analysis might be useful in pd stages before the advent of lewy pathology . because both alpha - synuclein and complexin-1 modulate vesicle release , our findings support presynaptic dysfunction as an early event in pd pathology . 
Introduction
Results
Overexpression of A53T-Alpha-Synuclein Modulates 
Overexpression of A53T-Alpha-Synuclein Leads to Elevated Complexin-1 Protein Levels in Mouse Midbrain/Brainstem
Deficiency of Alpha-Synuclein Also Modulates the Complexin-1 Levels in Mouse Midbrain/Brainstem
Discussion
Materials and Methods