Cellular regulation of the dopamine transporter

Jacob Eriksen

Cellular regulation of the dopamine transporter

Jacob Eriksen

645 Downloads (Pure)

Abstract

Dopamintransporteren er ansvarlig for genoptag af dopamin fra den synaptiske kløft og er det primære mål for virkningen af udbredte psykostimulantia så som kokain og amfetamin. Selvom DAT spiller en central rolle for virkningen af psykostimulantia, så er viden om fordeling og 'trafficking' af DAT i endogene systemer begrænset. I nærværende studie har vi brugt fluorescensmærkede kokainanaloger med høj affinitet for DAT. Disse kokainanaloger muliggør visualisering af DAT i levende primærkulturer af dopaminerge kulturer. I de dopaminerge nerveceller var DAT ensartet fordelt i plasma membranen både i soma og i nervecellens udløbere. FRAP (fluoresecens recovery after photobleaching) eksperimenter viste bidirektionel bevægelse af DAT, og desuden var der evidens for meget mobil DAT i både udløbere og varikositeter. DAT udviste konstitutiv endocytose og blev ophobet i vesikulære strukturer, der muligvis repræsenterer en intracellulær pulje af DAT. Den konstitutive endocytose kunne blokeres af lentiviral medieret ekspression af dominant negativ dynamin 1, og konstitutiv internaliseret DAT co-lokaliserede til dels med både EGFP-Rab5 og transferrin receptoren. Overraskende var DAT internalisering ikke påvirket af protein kinase C (PKC) aktivitet i vores primærkulturer af dopaminerge nerveceller. Hverken stimulering af PKC med PMA eller ved at hæmme PKC med GF109203X påvirkede internaliseringen eller dopaminoptag. Disse resultater er forskellige fra observationer i cellelinier, som exogent udtrykker DAT og sætter spørgsmålstegn ved paradigmet om PKC-medieret regulering af DAT i dopaminerge nerveceller. For yderligere at karakterisere den konstitutive internalisering, som vi observerede i de dopaminerge neuroner anvendte vi flere forskellige strategier til at detektere DAT i både cellelinier og dopaminerge nerveceller. For at undersøge internalisering af eksogent udtrykt DAT brugte vi et konstrukt med DAT N-terminalen fusioneret med den intracellulære del af enkelt transmembran domæne proteinet Tac for herved at skabe en ekstracellulær antistof-epitop. Når dette Tac-DAT fusionsprotein blev udtrykt i HEK293 celler udviste det funktionelle egenskaber svarende til vildtype transporteren og blev internaliseret i lighed med denne. I et ELISA eksperiment kunne vi kvantificere en konstitutiv tidsafhænigig DAT internaliserng i HEK293 og den medfølgende intracellulære akkumulering blev øget af leupeptin, en hæmmer af lysosomal degradering. Dette indikerer at Tac-DAT delvist blev sorteret til lysosomal degradering. For at videre undersøge denne hypotese udtrykte vi TacDAT i den dopaminerge cellelinie 1Rb3An27, og her undersøgte vi sorteringen af TacDAT v.h.a. co-udtryk af endosomale markører koblet til EGFP. Kvantificering viste at internaliseret TacDAT kolokaliserede med Rab7, som lokaliserer til sen endosomale vesikler, et forstadie til lysosomer. TacDAT kolokaliserede signifikant mere med Rab7 end Rab11, som lokaliserer til en anden type 'recycling' endosomer. For at undersøge om dette også var gældende for konstitutivt internaliseret DAT enodgent, udtrykte vi EGFP-Rab markørerne v.h.a. lentivirus i primærkulturer med dopaminerge nerveceller. Ved hjælp af kokain-analogen, JHC 1-64 observerede vi at konstitutivt internaliseret DAT colokaliserede med Rab7 og ikke Rab11. Desuden var der en udpræget colokalisering mellem DAT og den lysosomale markør LysoTracker. Vi foreslår derfor at konstitutivt internaliseret DAT sorteres til degradering via rab7 positive vesikler til lysosomer både i cellelinier og primærkulturer med dopaminerge nerveceller, som udtrykker DAT endogent. Samlet giver resultaterne et billede af en dynamisk opretholdelse DAT homøostase i dopaminerge nerveceller.

Originalsprog	Engelsk

Antal sider	196
Status	Udgivet - 2010

Adgang til dokumentet

PhD Thesis Jacob EriksenForlagets udgivne version, 16,5 MB

Citationsformater

@phdthesis{a3ae1860699a11df928f000ea68e967b,

title = "Cellular regulation of the dopamine transporter",

abstract = "The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft and is a target for widely abused psychostimulants such as cocaine and amphetamine. Nonetheless, little is known about the cellular distribution and trafficking of natively expressed DAT. DAT and its trafficking was visualized in cultured live midbrain dopaminergic (DA) neurons using novel fluorescently tagged cocaine analogs. The ligands enabled specific labelling and visualization of DAT in live neurons. In the DA neurons, DAT was uniformly distributed in the plasma membrane of the soma, the neuronal extensions, and varicosities along these extensions. Moreover, DAT was constitutively internalized into vesicular structures and the internalization was blocked by lentiviralmediated expression of dominant-negative dynamin. DAT internalization and function was not affected by activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or by inhibition with staurosporine or GF109203X. These data are in contrast to findings for DAT in transfected heterologous cells and challenge the paradigm that trafficking and cellular distribution of endogenous DAT is subject to regulation by PKC. To further characterize the intracellular pool of constitutively endocytosed DAT several strategies were employed in both cell lines and cultured DA neurons. First, to investigate the constitutive trafficking of heterologously expressed DAT we fused the N-terminus of DAT to the intracellular tail of the single-membrane spanning protein Tac, thereby creating an extracellular antibody epitope. Upon expression in HEK293 cells this TacDAT fusion protein displayed functional properties similar to the wild type transporter. In an ELISA based internalization assay, TacDAT intracellular accumulation was increased by inhibitors of lysosomal degradation and moreover TacDAT colocalized with the late endosomal marker Rab7. In the DA cell line 1Rb3An27 TacDAT also co-localized with EGFP-Rab7 and not with the recycling endosomal marker EGFP-Rab11. To assess whether sorting to late endosomes/lysosomes was a property also inherent to natively expressed transporter, DAT was visualized directly in cultured DA neurons using the fluorescent cocaine analog JHC 1-64. These data showed pronounced colocalization upon constitutive internalization with Lysotracker, a late endosomal/lysosomal marker; however only little cololization was observed with Alexa488-conjugated transferrin. Additionally, when expressing EGFP-Rab7 with lentivirus in cultured DA neurons a marked co-localization between JHC 1-64 and EGFP-Rab7 was observed. The data suggest that constitutively internalized DAT primarily is targeted to late endosomes and lysosomes both in heterologous cells and in cultured DA neurons.DAT has been shown to be regulated by the dopamine D2 receptor (D2R), the primary target foranti-psychotics, through a direct interaction. D2R is among other places expressed as an autoreceptor in DA neurons. Transient over-expression of DAT with D2R in HEK293 cells reduced surface expression of D2R while DAT surface levels remained unaffected. Furthermore {\ss}-Arrestin2 recruitment to D2R was compromised and D2R-mediated inhibition of cAMP accumulation was reduced as a consequence of the reduced surface level expression of D2R. The effects of DAT on D2R surface expression and signalling was not unique for DAT and D2R. Thus, transient coexpression of {\ss}2AR with DAT resulted in reduced surface levels of {\ss}2AR. In addition, coexpression of the serotonin transporter with D2R appeared to modulate the {\ss}-Arrestin2 recruitment to D2R and the D2R-mediated inhibition of cAMP accumulation in a manner similar to coexpression of DAT. The data suggest that DAT affect D2R function and expression with no apparent specificity.",

author = "Jacob Eriksen",

year = "2010",

language = "English",

}

TY - BOOK

T1 - Cellular regulation of the dopamine transporter

AU - Eriksen, Jacob

PY - 2010

Y1 - 2010

N2 - The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft and is a target for widely abused psychostimulants such as cocaine and amphetamine. Nonetheless, little is known about the cellular distribution and trafficking of natively expressed DAT. DAT and its trafficking was visualized in cultured live midbrain dopaminergic (DA) neurons using novel fluorescently tagged cocaine analogs. The ligands enabled specific labelling and visualization of DAT in live neurons. In the DA neurons, DAT was uniformly distributed in the plasma membrane of the soma, the neuronal extensions, and varicosities along these extensions. Moreover, DAT was constitutively internalized into vesicular structures and the internalization was blocked by lentiviralmediated expression of dominant-negative dynamin. DAT internalization and function was not affected by activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or by inhibition with staurosporine or GF109203X. These data are in contrast to findings for DAT in transfected heterologous cells and challenge the paradigm that trafficking and cellular distribution of endogenous DAT is subject to regulation by PKC. To further characterize the intracellular pool of constitutively endocytosed DAT several strategies were employed in both cell lines and cultured DA neurons. First, to investigate the constitutive trafficking of heterologously expressed DAT we fused the N-terminus of DAT to the intracellular tail of the single-membrane spanning protein Tac, thereby creating an extracellular antibody epitope. Upon expression in HEK293 cells this TacDAT fusion protein displayed functional properties similar to the wild type transporter. In an ELISA based internalization assay, TacDAT intracellular accumulation was increased by inhibitors of lysosomal degradation and moreover TacDAT colocalized with the late endosomal marker Rab7. In the DA cell line 1Rb3An27 TacDAT also co-localized with EGFP-Rab7 and not with the recycling endosomal marker EGFP-Rab11. To assess whether sorting to late endosomes/lysosomes was a property also inherent to natively expressed transporter, DAT was visualized directly in cultured DA neurons using the fluorescent cocaine analog JHC 1-64. These data showed pronounced colocalization upon constitutive internalization with Lysotracker, a late endosomal/lysosomal marker; however only little cololization was observed with Alexa488-conjugated transferrin. Additionally, when expressing EGFP-Rab7 with lentivirus in cultured DA neurons a marked co-localization between JHC 1-64 and EGFP-Rab7 was observed. The data suggest that constitutively internalized DAT primarily is targeted to late endosomes and lysosomes both in heterologous cells and in cultured DA neurons.DAT has been shown to be regulated by the dopamine D2 receptor (D2R), the primary target foranti-psychotics, through a direct interaction. D2R is among other places expressed as an autoreceptor in DA neurons. Transient over-expression of DAT with D2R in HEK293 cells reduced surface expression of D2R while DAT surface levels remained unaffected. Furthermore ß-Arrestin2 recruitment to D2R was compromised and D2R-mediated inhibition of cAMP accumulation was reduced as a consequence of the reduced surface level expression of D2R. The effects of DAT on D2R surface expression and signalling was not unique for DAT and D2R. Thus, transient coexpression of ß2AR with DAT resulted in reduced surface levels of ß2AR. In addition, coexpression of the serotonin transporter with D2R appeared to modulate the ß-Arrestin2 recruitment to D2R and the D2R-mediated inhibition of cAMP accumulation in a manner similar to coexpression of DAT. The data suggest that DAT affect D2R function and expression with no apparent specificity.

AB - The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft and is a target for widely abused psychostimulants such as cocaine and amphetamine. Nonetheless, little is known about the cellular distribution and trafficking of natively expressed DAT. DAT and its trafficking was visualized in cultured live midbrain dopaminergic (DA) neurons using novel fluorescently tagged cocaine analogs. The ligands enabled specific labelling and visualization of DAT in live neurons. In the DA neurons, DAT was uniformly distributed in the plasma membrane of the soma, the neuronal extensions, and varicosities along these extensions. Moreover, DAT was constitutively internalized into vesicular structures and the internalization was blocked by lentiviralmediated expression of dominant-negative dynamin. DAT internalization and function was not affected by activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or by inhibition with staurosporine or GF109203X. These data are in contrast to findings for DAT in transfected heterologous cells and challenge the paradigm that trafficking and cellular distribution of endogenous DAT is subject to regulation by PKC. To further characterize the intracellular pool of constitutively endocytosed DAT several strategies were employed in both cell lines and cultured DA neurons. First, to investigate the constitutive trafficking of heterologously expressed DAT we fused the N-terminus of DAT to the intracellular tail of the single-membrane spanning protein Tac, thereby creating an extracellular antibody epitope. Upon expression in HEK293 cells this TacDAT fusion protein displayed functional properties similar to the wild type transporter. In an ELISA based internalization assay, TacDAT intracellular accumulation was increased by inhibitors of lysosomal degradation and moreover TacDAT colocalized with the late endosomal marker Rab7. In the DA cell line 1Rb3An27 TacDAT also co-localized with EGFP-Rab7 and not with the recycling endosomal marker EGFP-Rab11. To assess whether sorting to late endosomes/lysosomes was a property also inherent to natively expressed transporter, DAT was visualized directly in cultured DA neurons using the fluorescent cocaine analog JHC 1-64. These data showed pronounced colocalization upon constitutive internalization with Lysotracker, a late endosomal/lysosomal marker; however only little cololization was observed with Alexa488-conjugated transferrin. Additionally, when expressing EGFP-Rab7 with lentivirus in cultured DA neurons a marked co-localization between JHC 1-64 and EGFP-Rab7 was observed. The data suggest that constitutively internalized DAT primarily is targeted to late endosomes and lysosomes both in heterologous cells and in cultured DA neurons.DAT has been shown to be regulated by the dopamine D2 receptor (D2R), the primary target foranti-psychotics, through a direct interaction. D2R is among other places expressed as an autoreceptor in DA neurons. Transient over-expression of DAT with D2R in HEK293 cells reduced surface expression of D2R while DAT surface levels remained unaffected. Furthermore ß-Arrestin2 recruitment to D2R was compromised and D2R-mediated inhibition of cAMP accumulation was reduced as a consequence of the reduced surface level expression of D2R. The effects of DAT on D2R surface expression and signalling was not unique for DAT and D2R. Thus, transient coexpression of ß2AR with DAT resulted in reduced surface levels of ß2AR. In addition, coexpression of the serotonin transporter with D2R appeared to modulate the ß-Arrestin2 recruitment to D2R and the D2R-mediated inhibition of cAMP accumulation in a manner similar to coexpression of DAT. The data suggest that DAT affect D2R function and expression with no apparent specificity.

M3 - Ph.D. thesis

BT - Cellular regulation of the dopamine transporter

ER -

Cellular regulation of the dopamine transporter

Abstract

Adgang til dokumentet

Fingeraftryk

Citationsformater