Tài liệu Báo cáo khoa học: P25a ⁄ TPPP expression increases plasma membrane presentation of the

P25a ⁄ TPPP expression increases plasma membrane

presentation of the dopamine transporter and enhances

cellular sensitivity to dopamine toxicity

Anja W. Fjorback1,2,*, Sabrina Sundbye2

, Justus C. Da¨ chsel2,, Steffen Sinning1

, Ove Wiborg1

and Poul H. Jensen2

1 Centre for Psychiatric Research, Aarhus University Hospital, Denmark

2 Department of Medical Biochemistry, Aarhus University, Denmark

Keywords

dopamine transporter; p25a; Parkinsons

disease; toxicity; TPPP; tubulin

polymerization promoting protein

Correspondence

P.H. Jensen, Department of Medical

Biochemistry, Aarhus University, Ole

Worms Alle 1170, DK-8000 Aarhus C,

Denmark

Fax: +45 86131160

Tel: +45 89422856

E-mail: [email protected]

*Present address

Stereology and EM Research Laboratory,

Aarhus University, Denmark

Present address

Division of Neurogenetics, Department of

Neuroscience, Mayo Clinic Florida,

Jacksonville, FL 32224, USA

(Received 21 June 2010, revised 1

November 2010, accepted 20 November

2010)

doi:10.1111/j.1742-4658.2010.07970.x

Parkinson’s disease is characterized by preferential degeneration of the

dopamine-producing neurons of the brain stem substantia nigra. Imbal￾ances between mechanisms governing dopamine transport across the

plasma membrane and cellular storage vesicles increase the level of toxic

pro-oxidative cytosolic dopamine. The microtubule-stabilizing protein p25a

accumulates in dopaminergic neurons in Parkinson’s disease. We hypothe￾sized that p25a modulates the subcellular localization of the dopamine

transporter via effects on sorting vesicles, and thereby indirectly affects its

cellular activity. Here we show that co-expression of the dopamine trans￾porter with p25a in HEK-293-MSR cells increases dopamine uptake via

increased plasma membrane presentation of the transporter. No direct

interaction between p25a and the dopamine transporter was demonstrated,

but they co-fractionated during subcellular fractionation of brain tissue

from striatum, and direct binding of p25a peptides to brain vesicles was

demonstrated. Truncations of the p25a peptide revealed that the require￾ment for stimulating dopamine uptake is located in the central core and

were similar to those required for vesicle binding. Co-expression of p25a

and the dopamine transporter in HEK-293-MSR cells sensitized them to

the toxicity of extracellular dopamine. Neuronal expression of p25a thus

holds the potential to sensitize the cells toward dopamine and toxins

carried by the dopamine transporter.

Structured digital abstract

l MINT-8055798: DAT (uniprotkb:Q01959) and p25 alpha (uniprotkb:O94811) colocalize

(MI:0403) by fluorescence microscopy (MI:0416)

l MINT-8054201: p25 alpha (uniprotkb:B1Q0K1), bip (refseq:GI:194033595), Synaptophysin

(uniprotkb:Q62277), Alpha-synuclein (uniprotkb:Q3I5G7) and DAT (uniprotkb:C6KE31)

colocalize (MI:0403) by cosedimentation in solution (MI:0028)

l MINT-8055878: Synaptophysin (uniprotkb:Q62277), bip (refseq:GI:194033595) and p25-alpha

(uniprotkb:O94811) colocalize (MI:0403) by cosedimentation through density gradient

(MI:0029)

Abbreviations

a-syn, a-synuclein; DA, dopamine; DAT, dopamine transporter; NET, norepinephrine transporter; PBSCM, phosphate-buffered saline

supplemented with Ca and Mg; PD, Parkinson’s disease; SERT, serotonin transporter; VMAT-2, vesicle monoamine transporter-2.

FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 493