Patent Application: US-59210605-A

Abstract:
modulation of the activity of serum and glucocorticoid inducible kinases to restore glutamate receptor activity . also disclosed are methods and compounds useful for the detection and treatment of neuropsychiatric disorders .

Description:
glutamate receptors activate a multitude of biochemical pathways in postsynaptic neurons eventually leading to postsynaptic neuronal plasticity thus an important aspect of the current invention is the teaching how the glutamate receptors are regulated . the study of regulation of glur1 required coexpression together with the various sgk isoforms . for testing sgk1 , sgk2 or sgk3 have been expressed together with the ampa receptor subunit glur1 in xenopus oocytes . a non - desensitizing glur1 mutant , glur1 ( l479y ) ( y . stern - bach , s . russo , m neumann , c . rosenmund , neuron 21 , 907 , 1998 ), has been used for the experiments . as illustrated in fig1 , the protein membrane abundance of glur1 is significantly increased in xenopus oocytes expressing glur1 together with sgk3 as compared to the glur1 protein abundance in oocytes expressing glur1 alone . glur1 protein abundance tended to be higher following coexpression of sgk2 , while coexpression of sgk1 was without effect . the protein abundance was paralleled by similar effects on glutamate - induced currents ( fig2 ). the glutamate - induced currents were significantly larger in xenopus oocytes expressing glur1 together with sgk3 than in xenopus oocytes expressing glur1 alone . the glutamate - induced currents in xenopus oocytes coexpressing sgk2 were significantly smaller than those in sgk3 - expressing oocytes , but significantly larger than currents in xenopus oocytes expressing glur1 alone . coexpression of sgk1 did not significantly modify glur1 - mediated currents . the present observations revealed a novel mechanism in the regulation of the glur1 subunit of ampa receptors . the delivery of glur1 to the neuronal surface is regulated by activation of nmda receptors , leading to ca 2 + entry ( m . sheng , m . j . kim , science 298 , 776 , 2002 ) with subsequent activation of pl3 - kinase ( m . s . perkinton , j . k . ip , g . l . wood , a . j . crossthwaite , r . j . williams , j . neurochem . 80 , 239 , 2002 ). activation of pl3 - kinase triggers a signaling cascade eventually leading to activation of sgk3 , which then enhances the protein abundance of glur1 in the cell membrane . sgk3 leads to a stabilized glur1 in the membrane thus preventing its retrieval and subsequent degradation and / or enhances trafficking of protein to the cell membrane . therefore the present observations describes for the first time that sgk2 and sgk3 substantially contributes to the fine tuning of glur1 abundance . according to the present findings sgk3 is expected to participate in glur1 - dependent neuronal function . glur1 is dominant over glur2 in heterodimeric glur1 - glur2 receptors ( y . hayashi , et al ., science 287 , 2262 , 2000 ; s . shi , y . hayashi , j . a . esteban , r . malinow , cell 105 , 331 , 2001 ), is required for hippocampal ca1 long - term potentiation ( d . zamanillo , et al ., science 284 , 1805 , 1999 ), and participates in the generation of spatial memory ( h . k . lee , et al ., cell 112 , 631 , 2003 ; d . reisel , et al ., nat . neurosci . 5 , 868 , 2002 ). to test for regulation of glur3 by the sgk isoforms the ampa receptor subunit glur3 was expressed the in xenopus oocytes with or without coexpression of either sgk1 , sgk2 or sgk3 . glutamate - induced currents were significantly smaller in xenopus oocytes ( fig5 ) expressing glur3 together with sgk2 than in xenopus oocytes expressing glur3 alone while coexpression with the related protein kinase b ( pkb ) was without significant effect . sgk1 and sgk3 similarly reduced the current amplitude but were less effective than sgk2 . administration of dexamethasone , a known modulator of sgk activity , for 8 or 20 days led to a significant increase of glur6 protein abundance in the mouse hippocampus ca3 neurons ( fig6 ) as seen in brain slices stained with glur6 polyclonal antibody . staining hippocampus ca3 neurons with map2 antibody , which is a marker for synaptic sites , identified enhanced glur6 staining at synapses . therefore , it was concluded that glur6 abundance is enhanced by dexamethasone at synaptic sites in hippocampal ca3 neurons . however , it cannot be distinguished between pre - or postsynaptic expression of glur6 . gfap , which specifically stains astrocytes , revealed that glur6 abundance in dexamathasone treated animals is not enhanced at astrocytes compared to control animals ( fig6 ). this result is consistent with predictions based on in situ hybridisation studies which have shown that sgk1 is not expressed in astrocytes ( waerntges et al .). to test for a functional link between sgk1 and glur6 , the rat ka receptor subunit glur6 was expressed in xenopus oocytes with or without coexpression of either sgk1 , sgk2 or sgk3 . as illustrated in fig4 , the protein abundance of glur6 is significantly enhanced in xenopus oocytes expressing glur6 together with sgk1 as compared to the glur6 protein abundance in oocytes expressing glur6 alone . a smaller but still statistically significant effect on glur6 protein abundance was observed following coexpression of sgk2 or sgk3 , while coexpression with the related protein kinase b ( pkb ) was without significant effect . similar to protein abundance , glutamate - induced current was significantly larger in xenopus oocytes expressing glur6 together with sgk1 than in xenopus oocytes expressing glur6 alone as shown in fig3 . again , sgk2 and sgk3 similarly stimulated the current but were significantly less effective than sgk1 . the present observations reveal a novel mechanism in the regulation of the glur6 subunit of ka receptors . the kainate receptors assembled with the glur6 subunit are important for the sensitivity of ca3 and ca1 pyramidal neurons to kainate and domoate ( bureau et al . 653 - 63 ). glur6 is unlikely to be the immediate target protein for sgk1 because glur6 does not contain the sgk1 recognition site rxrxxs / t . however , it can not be excluded that sgk1 recognizes other sites than this known amino acid sequence . the membrane protein stargazin has been shown to be critical for guiding ampa receptors to the cell surface and for targeting them specifically to postsynaptic sites . stargazin contains the sgk1 recognition site . however , it has been recently published that kars are not regulated by stargazin ( chen 2003 ). therefore , it is not expected that sgk1 regulates glur6 via stargazin which we confirmed by coinjection experiments in oocytes ( data not shown ). the inventive regulatory mechanism involving the new identified kinases is a powerful regulator of glur6 . sgk1 enhances the abundance of glur6 in the plasma membrane and increases glur6 mediated glutamate - induced currents . thus , sgk1 participates in the regulation of kainate receptor trafficking , synaptic plasticity and neuronal excitability . fig1 : increase of glur1 subunit protein abundance in the cell membrane of xenopus oocytes coexpressing sgk . ( a ) representative western blot . for the detection of glur1 , primary rabbit immuno affinity - purified anti - glur1 antibody ( 1 μg / μl , upstate ) was used . for the detection of β - tubulin , primary mouse monoclonal anti - β - tubulin antibody ( 1 : 250 , santa cruz ) was used . glur1 protein has an apparent molecular weight of ˜ 105 kda . ( b ) bar graph showing relative abundance of glur1 plasma membrane protein . the band intensities were quantified by arithmetic analysis using the software scion image . the values of three different blots from different batches were used for the statistical analysis . significant change ( p & lt ; 0 . 05 ) is indicated by 1 . fig2 : increase in glur1 currents by sgk2 and sgk3 isoforms but not by sgk1 and pkb . representative ( a ) current traces measured in xenopus oocytes in response to superfusion with 300 μm glutamate in nd96 ringer solution . oocytes were injected with glur1 crna ( 4 ng / oocyte ) or together with sgk crna ( 6 ng / oocyte ) ( b ) glur1 current amplitudes in oocytes expressing glur1 ( l479y )+ depc — h2o , glur1 ( l479y )+ sgk1 , glur1 ( l479y )+ sgk2 , glur1 ( l479y )+ sgk3 and glur1 ( l479y )+ pkb normalized to the glur1 + depc — h2o currents . horizontal scale bars indicate 5 sec and vertical scale bars represent 1 μa . numbers of oocytes are shown in parenthesis and significant changes ( p & lt ; 0 . 001 ) are indicated by ***. fig3 : increase in glur6 currents by sgk isoforms but not by pkb . ( a ) representative current traces measured in xenopus oocytes in response to superfusion with 300 μm glutamate . all currents were measured at 70 mv and after pretreatment of oocytes with cona to minimize desensitization . ( b ) glur6 current amplitudes in oocytes expressing glur6 + depc — h 2 o ( n = 20 ), glur6 + sgk1 ( n = 12 ), glur6 + sgk2 ( n = 10 ), glur6 + sgk3 ( n = 9 ) and glur6 + sgk1 ( n = 7 ) were measured and are shown normalized to the glur6 + depc — h 2 o currents . significant changes upon significance levels of p = 0 . 001 (***), p = 0 . 01 (**) and p = 0 . 05 (*) are indicated . fig4 : western blot demonstrating sgk - regulated protein expression of the glur6 subunit . plasma membrane protein was labeled with biotinyl - cona , solubilized , and then streptavidin - precipitated . samples including controls from uninjected oocytes were separated on a sds gel , western - blotted and probed with a immunoaffinity purified antibody directed against a 16 amino acid fragment of an c - terminus of glur6 ( upstate ). glur6 protein has an apparent molecular weight of ˜ 119 kda ( i ). fig5 : inhibition of glur3 mediated currents by co - expression sgk2 . ( a ) representative current traces measured in xenopus oocytes in response to superfusion with 300 μm glutamate . all currents were measured at 70 mv and after pretreatment of oocytes with cona to minimize desensitization . ( b ) glur3 current amplitudes in oocytes expressing glur3 + depc — h 2 o ( n = 20 ), glur3 + sgk1 ( n = 12 ), glur3 + sgk3 ( n = 10 ), glur3 + sgk1 ( n = 9 ), glur3 + sgk3 ( n = 9 ) and glur3 + sgk1 ( n = 7 ) were measured and are shown normalized to the glur3 + depc — h 2 o currents . significant changes upon significance levels of p = 0 . 001 (***), p = 0 . 01 (**) and p = 0 . 05 (*) are indicated . fig6 : expression of glur6 in hippocampus of dexamethasone treated and control mice . administration of dexamethasone for 8 or 20 days led to a significant increase of glur6 protein abundance in the mouse hippocampus ca3 neurons ( b ) as seen in brain slices stained with glur6 polyclonal antibody . staining hippocampus ca3 neurons with map2 antibody , which is a marker for synaptic sites , identified enhanced glur6 staining at synapses . oocytes of stages v - vi were surgically removed from the ovaries of xenopus laevis as described elsewhere ( seebohm , sanguinetti , pusch , 2003 ). oocytes were injected with either 4 ng of glur1 or glur3 or glur6 crna or with or without 6 ng sgk1 or sgk2 or sgk3 or pkb crna using a nanoliter 2000 injector ( wpi inc ., florida , usa ). standard two - electrode voltage clamp recordings were performed 5 - 8 days after crna injection with a turbotec 03 amplifier ( npi , tamm , germany ) and an interface digidata 1322a from axon instruments . data analyses were done with pclamp 9 . 0 / clampfit 9 . 0 software ( axon inc . ), and origin 6 . 0 software ( microcal ). agonist solutions were prepared in nd - 96 buffer ( in mm , nacl , 96 ; cacl 2 , 1 , 8 ; kcl , 2 , 0 ; mgcl 2 , 1 , 0 and hepes - naoh , 5 , ph 7 . 2 with naoh ). current and voltage electrodes were filled with 3 m kcl and had resistances of 0 . 5 - 1 . 5 mω . oocytes were held at − 70 mv and agonist ( 300 μm glutamate ) was applied by superfusion for ˜ 10 s at a flow rate of 10 - 14 ml / min . prior to agonist application , the oocyte was incubated for 8 min in concanavalin a to prevent desensitization . to identify the fraction of receptor protein inserted in the plasma membrane , surface proteins were tagged with biotinylated cona and isolated by streptavidin / sepharose - mediated precipitation of the biotinyl - cona / protein complex . briefly , intact oocytes were incubated in 10 μm biotinyl - cona ( sigma , münchen , germany ) for 30 min at room temperature . after five 10 - min washes in normal frog ringer , intact oocytes were homogenized with a teflon pestle in h - buffer ( 20 μl / oocyte ; 100 mm nacl , 20 mm tris - hcl , ph 7 . 4 , 1 % triton x - 100 , 1 mm phenylmethylsulphonyl fluoride plus a mixture of proteinase inhibitors ( complete ™ tablets , boehringer )) and were kept at 4 ° c . for 1 h on a rotator . after centrifugation for 60 s at 16000 × g , the supernatants were supplemented with 20 μl washed streptavidin - sepharose beads ( sigma , münchen , germany ) and incubated at 4 ° c . for 3 h on the rotator . the streptavidin - sepharose beads were then pelleted by a 120 s spin at 1600 × g and washed three times in h - buffer . the final pellets were boiled in 40 μl sodium dodecylsulphate - polyacrylamide gel electrophoresis ( sds - page ) loading buffer ( 0 . 8 m β - mercaptoethanol , 6 % sds , 20 % glycerol , 25 mm tris - hcl , ph 6 . 8 , 0 . 1 % bromphenol blue ). measured using an elisa kit ( mercodia , uppsala , sweden ). proteins from homogenized oocytes were separated by sds electrophoresis and transferred to nitrocellulose filters . blots were blocked in 1 × pbs containing 5 % milk powder for at least 1 hour at room temperature . for the detection of glur1 , glur3 or glur6 , primary rabbit immunoaffinity purified glur1 , glur3 or glur6 antibody ( 1 μg / μl , upstate ) and secondary horseradish peroxidase - conjugated donkey anti - rabbit antibody ( 1 : 1000 dilution , amersham bioscience ) were used . for verification of protein leves , ponceau red staining was performed . 4 . 1 . compounds of the general formula i and pharmaceutical useful derivates , salts , solutions and stereoisomeres thereof including mixtures . r 1 , r 5 is either h , oh , oa , oac or methyl , r 2 , r 3 , r 4 , r 6 , r 7 , r 8 , r 9 , r 10 is either h , oh , oa , oac , ocf 3 , hai , no 2 , cf 3 , a , cn , oso 2 ch 3 , so 2 ch 3 , nh 2 or cooh , compound according to formula i selected from the following group of compounds : ( 4 - fluor - phenyl )- acidic acid -( 4 - hydroxy - 2 - methoxy - benzyliden )- hydrazid , ( 2 - chlor - 4 - fluor - phenyl )- acidic acid -( 4 - hydroxy - 2 - methoxy - benzyliden )- hydrazid , ( 3 - fluor - phenyl )- acidic acid -( 4 - hydroxy - 2 - methoxy - benzyliden )- hydrazid , ( 3 - methoxy - phenyl )- acidic acid -( 4 - hydroxy - benzyliden )- hydrazid , ( 3 - methoxy - phenyl )- acidic acid -( 4 - hydroxy - 2 , 6 - dimethyl - benzyliden )- hydrazid , ( 3 - methoxy - phenyl )- acidic acid -( 3 - fluor - 4 - hydroxy - benzyliden )- hydrazid , ( 3 - methoxy - phenyl )- acidic acid -[ 1 -( 4 - hydroxy - 2 - methoxy - phenyl )- ethyliden ]- hydrazid , ( 3 - methylsulfonyloxy - phenyl )- acidic acid -( 4 - hydroxy - 2 - methoxy - benzyliden )- hydrazid , ( 3 , 5 - dihydroxy - phenyl )- acidic acid -( 4 - hydroxy - 2 - methoxy - benzyliden )- hydrazid , ( 3 - fluor - phenyl )- acidic acid -( 3 - fluor - 4 - hydroxy - benzyliden )- hydrazid , ( 3 - methoxy - phenyl )- acidic acid -( 4 - acetoxy - 2 - methoxy - benzyliden )- hydrazid , ( 3 - trifluormethyl - phenyl )- acidic acid -( 4 - hydroxy - 2 - methoxy - benzyliden )- hydrazid , 3 -( 3 - methoxy - phenyl )- propionsäure -( 4 - hydroxy - 2 - methoxy - benzyliden )- hydrazid , ( 3 - methoxy - phenyl )- acidic acid -( 2 , 4 - dihydroxy - benzyliden )- hydrazid , ( 3 - methoxy - phenoxy )- acidic acid -( 4 - hydroxy - 2 - methoxy - benzyliden )- hydrazid , ( 3 - nitro - phenyl )- acidic acid -( 4 - hydroxy - 2 - methoxy - benzyliden )- hydrazid , ( 3 - methoxy - phenyl )- acidic acid -( 5 - chlor - 2 - hydroxy - benzyliden )- hydrazid , ( 3 - methoxy - phenyl )- acidic acid -( 2 - hydroxy - 5 - nitro - benzyliden )- hydrazid , 2 - hydroxy - 2 - phenyl - acidic acid -( 4 - hydroxy - 2 - methoxy - benzyliden )- hydrazid , ( 3 - methoxy - phenyl )- acidic acid -( 2 - ethoxy - 4 - hydroxy - benzyliden )- hydrazid , ( 3 - brom - phenyl )- acidic acid -( 4 - hydroxy - 2 - methoxy - benzyliden )- hydrazid , ( 3 - methoxy - phenyl )- acidic acid -[ 1 -( 4 - hydroxy - phenyl )- ethyliden ]- hydrazid , ( 3 , 5 - difluor - phenyl )- acidic acid -( 4 - hydroxy - 2 - methoxy - benzyliden )- hydrazid , ( 3 - hydroxy - phenyl )- acidic acid -( 4 - hydroxy - 2 - methyl - benzyliden )- hydrazid , ( 3 - hydroxy - phenyl )- acidic acid -( 2 - ethoxy - 4 - hydroxy - benzyliden )- hydrazid , ( 3 - hydroxy - phenyl )- acidic acid -( 2 - methoxy - 4 - hydroxy - 6 - methyl - benzyliden )- hydrazid , ( 2 - fluor - phenyl )- acidic acid -( 2 - methoxy - 4 - hydroxy - benzyliden )- hydrazid 4 . 2 . compounds of the general formula ii and pharmaceutical useful derivates , salts , solutions and stereoisomeres thereof including mixtures . r 4 , r 5 is either h , a , oh , oa , alkenyl , alkinyl , no 2 , nh 2 , nha , na 2 , hai , cn , cooh , cooa , two groups selected from r 1 , r 2 , r 3 , r 4 , r 5 or as well — o — ch 2 — ch 2 —, — o — ch 2 — o — or — o — ch 2 — ch 2 — o —, r 6 , r 7 is either h , a , hal , oh , oa or cn , is a saturated or unsaturated heterocycle with 1 to 4 n -, o - and / or s - atoms , substituted by one or several hal , a , oa , cooa , cn or carbonyloxigen (═ o ) a alkyl with 1 to 10 c - atoms , wherein 1 - 7 h - atoms may be replaced by f and / or chlorine , compound according to formula ii selected from the following group of compounds : the nucleotide sequence defining intron 6 of facultative hypertensive patients is . . . aattacatt c gcaacccag . . . , whereas the nucleotide sequence representing a healthy population is . . . aattacatt t gcaacccag . . . . the sequences are available through accession number gi 2463200 position 2071 . the exon 8 sequences of facultative hypertensive patients are either homozygotic . . . tactga c ttcggact . . . or . . . tactgatttcggact . . . or heterozygotic . . . tactga c ttcggact . . . and . . . tactgatttcggact . . . . the sequences are available through accession number nm — 005627 . 2 , position 777 . a homozygotic individual with a tt nucleotide combination is protected even if simultaneously a cc single nucleotide polymorphism is presented in intron 6 . expression of glur6 in hippocampus of dexamethasone treated ice and control mice age and sex matched siblings of sv129j mice were used for this study . 2 - 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