familial danish dementia ( fdd ) is a neurodegenerative disease characterized by early cataracts , deafness , progressive ataxia , and dementia . neurofibrillary tangles ( nfts ) are the major histological finding in the hippocampus [ 13 ] . fdd is an autosomal dominant disease caused by the accumulation of the amyloidogenic c - terminal 34 amino acid peptide of bri2 . bri2 , a product of the itm2b / bri2 gene , is a type ii transmembrane protein of unknown function . physiologically , bri2 is cleaved at the c - terminus by furin endoprotease , producing a 23-amino acid soluble c - terminal fragment . fdd is caused by a decamer duplication in the 3 region of the itm2b / bri2 gene . this mutation produces a longer 34 amino acid c - terminal fragment that accumulates as amyloid [ 4 , 5 ] . fdd and alzheimer 's disease ( ad ) share neuropathological features , including amyloid deposition and neurodegeneration in the central nervous system [ 68 ] . the mature form of bri2 is able to reduce accumulation of the a peptide [ 912 ] while app catabolites , including a , were significantly increased in the brain of fdd patients . the interaction between these two amyloidogenic proteins ( bri2 and app ) can be relevant for the design of new therapeutic strategies for fdd and ad . to model fdd pathogenesis in animals , several mouse lines have been generated and analyzed so far , which include transgenic mice expressing wild - type   or mutant [ 16 , 17 ] bri2 proteins , as well as itm2b / bri2 gene knock - out mice   or itm2b / bri2 knock - in mutant mice . the transgenic mice overexpressing the fdd - associated mutation of human itm2b / bri2 partially recapitulated the main histopathological features of fdd ; they indeed developed cerebral amyloid angiopathy , parenchymal amyloid deposition , and neuroinflammation [ 14 , 16 ] . age - dependent deposition of danish amyloid peptide was also observed in the brain of a second transgenic model , with associated angiopathy and microhemorrhage , neuritic dystrophy , and neuroinflammation . in this model , deposition of the danish peptide also accelerated tau pathology in a double transgenic model with mutant itm2b / bri2 and tau ( p301s ) constructs . to study the pathogenesis of fdd in the absence of potential artifacts due to transgenic overexpression of the mutant protein , a refined mouse model has been recently generated , which is based on a knock - in approach where the exon 6 of the murine itm2b / bri2 gene was substituted for by a mutant sequence carrying the human fdd mutation . the analysis of fddki mouse model , carrying a mutant and a wild - type itm2b / bri2 allele , has shown that the danish mutation causes impaired synaptic plasticity and deficits in hippocampal memory , in the absence of cerebral deposits and lesions . a similar scenario applies to a familial british dementia ( fbd ) knock - in model , in which the dominant , mutant itm2b / bri2 allele responsible for fbd is associated to memory deficits and loss of bri2 function , without the histopathological alterations typical to neurodegenerative disorders . in agreement with the previously reported function of bri2 in the stabilization of app holoprotein [ 9 , 12 ] , fddki mice also show decreased bri2 protein levels and increased levels of app catabolites ; the latter evidence recapitulated the occurrence of increased app metabolites in the brain of an fdd patient . the functional link between itm2b / bri2 and app genes is also strongly supported by the genetic suppression of memory and synaptic dysfunctions in fddki mice by app haplodeficiency   or inhibition of app cleavage by -secretase . the functional association between itm2b / bri2 and app genes strongly supports the participation of the encoded proteins to synaptic dysfunction . accordingly , decreased levels of bri2/app complexes have been observed in the synaptic membranes of fddki mice . in this paper , we profiled the expression pattern of synaptosomal proteins in the fddki mouse model , via differential expression analysis based on two - dimensional differential in - gel electrophoresis ( 2d - dige ) in combination with tandem mass spectrometry . our validated results showed decreased representation of the synaptosomal scaffold protein psd95/dlg4 and of the dusp3 phosphatase in the fddki synaptosomes . mice were handled according to the ethical guidelines for treatment of laboratory animals of the albert einstein college of medicine . crude synaptosomal fractions containing both membrane and soluble synaptosomal proteins were obtained from three biological replicates each of wild - type ( c57bl/6j ) or fddki mice , as described in . briefly , mouse brain homogenates in hepes - sucrose buffer ( 20  mm hepes / naoh ph 7.4 , 1  mm edta , 1  mm egta , and 0.25  m sucrose ) supplemented with protease and phosphatase inhibitors ( wt / vol = 10  mg tissue/100  ml buffer ) were centrifuged at 800  g for 10  min , at 4c . the supernatant ( s1 , total brain extract ) was separated into supernatant ( s2 ) and pellet ( p2 ) by centrifugation at 9,200  g for 15  min , at 4c . p2 fractions , representing the crude synaptosomal fractions , were resuspended in 7  m urea , 2  m thiourea , 4% chaps to extract the whole synaptosomal content , then precipitated in acetone / methanol ( 8  :  1 , v  :  v ) for 16  h , at 20c , and finally recovered by centrifugation at 16,000  g for 30  min , at 4c . the synaptosomal proteins were resuspended in 7  m urea , 2  m thiourea , 4% chaps , and 30  mm tris - hcl ; protein concentration was determined by using the bradford method ( bio - rad ) . before labelling , the ph of the samples was adjusted to ph 8.5 with hcl solutions ; each labelling reaction was performed in a 10  l volume with 50  g of the synaptosomal proteins , in the presence of 400  pmol of cy2- , cy3- , or cy5-dyes ( minimal labelling dyes , ge healthcare ) . a dye - swapping strategy was used to label samples ; accordingly , two samples from wild - type synaptosomal extracts were labelled with cy3 , while the third was labelled with cy5 ; in a complementary manner , two samples from fddki extracts were labelled with cy5 , and the third was labelled with cy3 . three mixtures of the 6 samples ( 50  g each ) were also labelled with cy2 dye , as the internal standard required for the 2d - dige procedure . the labelling was performed for 30  min , at 0c , in the dark , and was chased by the addition of 1  mm lysine . three sample mixtures , made of appropriate cy3- and cy5-labeled pairs and a cy2-labeled control , were supplemented with 1% ( v / v ) ipg buffer , ph 310 nl ( ge healthcare ) , 1.4% ( v / v ) destreak reagent ( ge healthcare ) , and 0.2% ( w / v ) dtt to a final volume of 450  l in 7  m urea , 2  m thiourea , and 4% chaps . the mixtures ( 150  g of total protein content ) were used to passively hydrate the immobilized ph gradient ipg gel strips ( 24  cm , ph 310  nl ) for 16  h , at 20c . isoelectric focusing ( ief ) was carried out with an ipgphor ii apparatus ( ge healthcare ) up to 75,000  v / h , at 20c ( current limit set to 50  a per strip ) . the strips were equilibrated in 6  m urea , 2% sds , 20% glycerol , and 0.375  m tris - hcl ( ph 8.8 ) , for 15  min , in the dark , in the presence of 0.5% ( w  :  v ) dtt , and then in the presence of 4.5% ( w / v ) iodoacetamide in the same buffer , for additional 15  min . equilibrated ipg strips were transferred onto 12% polyacrylamide gels , within low - fluorescence glass plates ( ettan - dalt 6 system , ge healthcare ) . the second - dimension sds - page was performed by using a dalt ii electrophoresis unit ( ge healthcare ) at 2  w / gel for 12  h. gels were scanned with a typhoon 9400 variable mode imager ( ge healthcare ) , using appropriate excitation / emission wavelengths for cy2 ( 488/520  nm ) , cy3 ( 532/580  nm ) , and cy5 ( 633/670  nm ) . images were acquired in the image - quant software ( ge healthcare ) and analyzed by using the decyder 6.0 software ( ge healthcare ) . a decyder differential in - gel - analysis ( dia ) module was used for spot detection and pairwise comparison of each sample ( cy3 and cy5 ) to the cy2 , mixed standard present in each gel . the decyder biological variation analysis ( bva ) module was then used to simultaneously match all of the protein - spot maps from the gels , and to calculate average abundance ratios and p values across the triplicate sets of samples ( student 's t - test ) .   for preparative protein separations , 500  g of unlabeled protein samples from pooled , wild - type , and fddki synaptosomal samples were used for passive hydration of 24  cm strips for the first gel dimension ( ph 310  nl ipg strips , ge healthcare ) . after 2-de , gels were fixed and stained with syproruby fluorescent stain ( invitrogen ) . after spot matching with the master gel from the analytical step in the bva module of decyder software , a pick list was generated for spot picking by a robotic picker ( ettan spot picker , ge healthcare ) . proteins were in - gel reduced , s - alkylated , and digested with trypsin , as previously reported . protein digests were subjected to a desalting / concentration step on ziptipc18 pipette tips ( millipore corp . , bedford , ma , usa ) and then analyzed by nanolc - esi - lit - ms / ms using a ltq xl mass spectrometer ( thermofinnigan , usa ) equipped with proxeon nanospray source connected to an easy - nanolc ( proxeon , denmark ) [ 21 , 22 ] . peptide mixtures were separated on an easy c18 column ( 10    0.075  mm , 3  m ) ( proxeon ) using a gradient of acetonitrile containing 0.1% formic acid in aqueous 0.1% formic acid ; acetonitrile ramped from 5% to 35% over 15  min and from 35% to 95% over 2  min , at a flow rate of 300  nl / min . acquisition was controlled by a data - dependent product ion - scanning procedure over the three most abundant ions , enabling dynamic exclusion ( repeat count 2 and exclusion duration 1  min ) . the mass isolation window and collision energy were set to m / z 3 and 35% , respectively . mascot software package version 2.2.06 ( matrix science , uk )   was used to identify spots unambiguously from a nonredundant sequence database ( all taxa uniprotkb 2010/01/09 ) . nanolc - esi - lit - ms / ms data were searched by using a mass tolerance value of 2  da for precursor ion and 0.8  da for ms / ms fragments , trypsin as proteolytic enzyme , a missed cleavages maximum value of 2 and cys carbamidomethylation , and met oxidation as fixed and variable modification , respectively . candidates with more than 2 assigned peptides with an individual mascot score > 25 , both corresponding to p < 0.05 for a significant identification , were further evaluated by the comparison with their calculated mass and pi values , using the experimental values obtained from 2-de . search for functional interaction between proteins identified by ms analysis was performed using the string v. 8.3 database ( http://string83.embl.de/ ) . gene ontology classification of the identified proteins was performed through the web - accessible david ( v 6.7 ) annotation system ( http://david.abcc.ncifcrf.gov/home.jsp ) [ 25 , 26 ] . briefly , the identified proteins were converted into refseq - protein identifiers through the david gene i d conversion tool ; the new list was then submitted to functional annotation clustering . triplicate synaptosomal samples from wild - type and fddki mice were separated on 10% polyacrylamide gels by sds - page and then blotted on pvdf membranes ( ge healthcare ) . filters were blocked in pbs containing 5% nonfat dry milk and incubated with 1  :  250 dilutions of the different primary antibodies , obtained from santa cruz biotechnology , except for tubulin ( dm1a ; sigma ) antibody . appropriate secondary igg horseradish peroxidase conjugated was used as secondary antibody ( southern biotechnology ) ; bands were visualised by the ecl kit ( ge healthcare ) . in order to characterize the synaptosomal proteome of the available mouse model of fdd , we used the differential in - gel electrophoresis approach ( 2d - dige ) , which was applied to synaptosomal extracts from wild - type and fddki mice . indeed , three biological replicates of synaptosomal preparations from wild - type and fddki mice were generated for protein extraction and fluorescence labelling with cy - dyes . analysis of the 2d - dige images , according to the decyder bioinformatic software , allowed us to detect about 2,000 matched protein spots within the three gels . we performed a quantitative and statistical analysis under parameters defined as relative expression ratio in fddki versus wt mice of > 1.25 for protein spots upregulated and of < 1.25 for protein spots downregulated , with a p value < 0.05 . we found that twelve spots appeared to be deregulated ( circled in figure 1(a ) ) ; in particular , four protein spots were upregulated , whereas eight protein spots were downregulated , in fddki versus wt mice . in order to identify the differentially expressed proteins , the spots of interest were matched to the corresponding ones from a preparative gel stained with fluorescent stain syproruby ( yellow circles in figure 1(b ) ) , which were then individually excised , digested with trypsin , and finally identified by nanolc - esi - lit - ms / ms analysis , as detailed in methods .   table 1 summarizes the relative spot expression ratios in fddki versus wt mice as well as the mass spectrometry data for the corresponding identified proteins . in order to find relevant proteins among the multiple identifications obtained by proteomic analysis , we subjected the list of the 13 proteins from table 1 to bioinformatic analysis in the string database , which integrates interaction data from several bioinformatic sources and provides information about physical and functional , known and predicted interactions of genes and their products   ( figure 2 ) . string analysis showed that dlg4 protein is directly linked to 4 additional proteins , namely ppp3ca , hspa8 , atp6v1a , and gnb1 . the evidences responsible for those functional associations are mainly experimental ( purple line ) , and reflect protein - protein interactions [ 2729 ] . indeed , dlg4 , also known as postsynaptic density protein 95 ( psd95 ) , contributes to the organization of multiprotein complexes at the postsynaptic contact zone , via its multiple protein interaction domains . this molecular network is made of receptors ( i.e. , nmda and ampa receptors ) , small signalling proteins ( i.e. , ras - gap ) , cell adhesion and cytoskeletal proteins , as well as protein kinases ( i.e. , camkii ) , thus organizing receptors and signal transduction molecules at the synaptic contact zone [ 30 , 31 ] . on its own , gnb1 is also tightly connected to gnb2 ; indeed , the two proteins are the beta 1 and beta 2 components of heterotrimeric g protein complexes , respectively . these proteins share a high protein sequence identity ( > 90% ) in mouse , in fact they were coidentified in spot 2632 ( table 1 ) . additional associations involving the differentially expressed proteins in fddki mice involved the kinesin family member 5a ( kif5a ) , a microtubule - dependent motor protein involved in the axonal transport of neurofilament proteins and organelles , and the kinesin family member 5c ( kif5c ) , involved in organelle transport . interestingly , kif5a and an additional protein identified in this study in multiple deregulated spots ( spots 890 , 891 , 918 , and 957 ) , namely , dihydropyrimidinase - related protein 2 ( dpysl2 ) , were found as differentially expressed during a proteomic profiling of an ad - transgenic model with preplaque ( 2 months ) and plaque ( 24 months ) phenotypes . the david database , which analyzes gene or protein lists deriving from high - throughput experiments and systematically extracts biological meaning from them [ 25 , 26 ] , was also used to highlight the functional annotation clustering inside the potential protein network identified by our proteomic analysis . ( goterm_mf_all ; p = 6.2e  5 ) , which included 6 out of the 13 identified proteins ( atp6v1a , kif5a , kif5c , gnb1 , gnb2 , and hspa8 ; figure 2 , proteins inside the blue box ) . an additional cluster was associated to the  mapk signaling pathway  and included heat shock cognate 71  kda protein ( hspa8 ) and two phosphatases , namely , protein phosphatase 3 , catalytic subunit , alpha isoform ( ppp3ca ) , and dual specificity phosphatase 3 ( dusp3 ) ( kegg_pathway p = 6.2e  2 ) ( figure 2 , green box ) . these phosphatases , although at a lower statistical value , were also annotated with the atpase , h transporting , lysosomal v1 subunit a ( atp6v1a ) in the biological process  phosphate metabolic process ( goterm_bp_fat p = 2.7e  1 ) . differentially expressed proteins from 2d - dige analysis were mainly classified according to their functional annotation into  nucleoside - triphosphatase activity  or their involvement in we then selected some representative members of the two groups , namely kif5a and dpysl2 , together with ppp3ca and dusp3 , respectively , to validate our expression results by an alternative method , such as western blotting . an additional downregulated protein , namely dlg4/psd95 , which is a prominent scaffold protein in synaptosomal architecture / function , and is also associated with nucleoside - triphosphatase activity , was also assayed . western blotting analysis definitively demonstrated that dlg4/psd95 was slightly downregulated in synaptosomal extracts from fddki mice , in strong agreement with 2d - dige data ( expression ratio 1.27 , p = 0.025 ; see table 1 ) ( figure 3(a ) ) . nucleoside - triphosphatase activity ,  namely , kif5a and dpysl2 ( figure 3(a ) ) . alternative results were obtained for phosphatase members of the  mapk signaling cascade .  in fact , a good correlation between 2d - dige and western blotting analysis was observed for dusp3 , while contradictory results were obtained for ppp3ca ( figure 3(a ) ) . negative correlation data between the two analytical methods were easily explained on the basis of the differential expression of specific protein isoforms bearing posttranslational modifications . thus , post - transcriptional events should regulate abundance of specific protein isoforms in fddki synaptosomes . in this context , for example , after exocytosis for neurotransmitter release , the synaptic vesicles are retrieved by endocytosis to accomplish additional cycles of synaptic transmission . in the paradygm , several proteins involved in synaptic function are dephosphorylated , during synaptic vesicle endocytosis , and rephosphorylated after recycling to guarantee the maintenance of synaptic transmission . although global levels of this protein seemed unchanged in the fddki synaptosomes by western blotting , 2d - dige analysis suggested downregulation of a specific ppp3ca isoform . on the contrary , dusp3 downregulation in the fddki synaptosomes was definitively validated in the synaptosomal preparations , as a nongeneralized phenomenon in mouse brain . in fact , western blot analysis of dusp3 in crude brain lysates showed slightly increased protein levels in the unfractionated fddki samples ( figure 3(b ) ) . dual - specificity phosphatases constitute a heterogeneous group of enzymes with the ability to recognize either phospho - tyr or phospho - ser / thr substrates and act in the regulation of jnk- and erk - mediated pathways . at the present , no evidences have been published that support a dusp3 function in the nerve system and in synaptic functionality , although one can speculate the involvement of this phosphatase also in the central nerve system , given the relevant functions of the erk kinases in the signal transduction mechanisms in the brain . the results of this work outline a differential expression of selected proteins in the synaptosomes of fddki mice . eight protein spots were downregulated , while four protein spots were upregulated in the fddki synaptosomal extracts . western blot analysis on selected proteins , guided by the results of bioinformatic classification of the deregulated proteins , showed that two signalling proteins , namely , dlg4/psd95 and dusp3 , were actually downregulated in the available animal model of fdd . the first one ( dlg4/psd95 ) is a well - known scaffold protein in synaptic function , and its downrepresentation in the synaptosomes of fddki mice may impair synaptic transmission and activity . the second protein ( dusp3 ) , a know regulator of jnk- and erk - mediated pathways , is not as well studied in the central nerve system . however , the evidence provided may stimulate further studies at the phosphoproteomic and functional levels , to assess its synaptosomal targets , and to define the role of its downregulation in brain physiology and in the mechanisms associated with fdd and additional neurodegenerative disorders , including fbd and ad .
a dominant mutation in the itm2b / bri2 gene causes familial danish dementia ( fdd ) in humans . to model fdd in animal systems , 
 a knock - in approach was recently implemented in mice expressing a wild - type and mutant allele , which bears the fdd - associated mutation . 
 since these fddki mice show behavioural alterations and impaired synaptic function , we characterized their synaptosomal proteome via two - dimensional differential in - gel electrophoresis . 
 after identification by nanoliquid chromatography coupled to electrospray - linear ion trap tandem mass spectrometry , the differentially expressed proteins were classified according to their gene ontology descriptions and their predicted functional interactions . 
 the dlg4/psd95 scaffold protein and additional signalling proteins , including protein phosphatases , were revealed by string analysis as potential players in the altered synaptic function of fddki mice . 
 immunoblotting analysis finally demonstrated the actual downregulation of the synaptosomal scaffold protein dlg4/psd95 and of the dual - specificity phosphatase dusp3 in the synaptosomes of fddki mice .