Characterization and transplantation of two neuronal cell lines with dopaminergic properties

The dopamine transporter (DAT) mediates reuptake of released dopamine and is the target for psychostimulants, such as cocaine and amphetamine. DAT undergoes marked constitutive endocytosis, but little is known about the fate and sorting of the endocytosed transporter. To study DAT sorting in cells lines, we fused the one-transmembrane segment protein Tac to DAT, thereby generating a transporter (TacDAT) with an extracellular antibody epitope suited for trafficking studies. TacDAT was functional and endocytosed constitutively in HEK293 cells. According to an ELISA-based assay, TacDAT intracellular accumulation was increased by the lysosomal protease inhibitor leupeptin and by monensin, an inhibitor of lysosomal degradation and recycling. Monensin also reduced TacDAT surface expression consistent with partial recycling. In both HEK293 cells and in the dopaminergic cell line 1Rb3An27, constitutively internalized TacDAT displayed primary co-localization with the late endosomal marker Rab7, less co-localization with the "short loop" recycling marker Rab4, and little co-localization with the marker of "long loop" recycling endosomes, Rab11. Removal by mutation of N-terminal ubiquitination sites did not affect this sorting pattern. The sorting pattern was distinct from a bona fide recycling membrane protein, the ␤ 2 -adrenergic receptor, that co-localized primarily with Rab11 and Rab4. Constitutively internalized wild type DAT probed with the fluorescently tagged cocaine analogue JHC 1-64, exhibited the same co-localization pattern as TacDAT in 1Rb3An27 cells and in cultured midbrain dopaminergic neurons. We conclude that DAT is constitutively internalized and sorted in a ubiquitination-independent manner to late endosomes/ lysosomes and in part to a Rab4 positive short loop recycling pathway.

Section: Dat Tacdatsupporting

confidence: 72%

Postendocytic Sorting of Constitutively Internalized Dopamine Transporter in Cell Lines and Dopaminergic Neurons

Eriksen

Bjørn‐Yoshimoto

Jørgensen

et al. 2010

“…Our studies directly assess the effect of ␣-synuclein on DAT activity when ␣-synuclein is dialyzed into DA neuron (21, 37) or immortalized mesencephalic neuronal cells (IRB3AN27) stably overexpress human dopamine transporter (38,39,64) via the patch clamp electrode or when ␣-synuclein is transiently overexpressed. Dialysis of ␣-synuclein into the cell via the patch clamp electrode allows real time assessment of DAT activity before and after intracellular ␣-synuclein under identical conditions and, importantly, in the same cell.…”

Section: Discussionmentioning

confidence: 99%

α-Synuclein Stimulates a Dopamine Transporter-dependent Chloride Current and Modulates the Activity of the Transporter

Swant

Goodwin

North

et al. 2011

Background: Dopamine transporter (DAT) and ␣-synuclein interact, but the mode and mechanism of ␣-synuclein regulation of DAT activity are less known. Results: Elevated intracellular ␣-synuclein alters DAT-mediated currents and activity that are abrogated by DAT blocker and are absent with heat-inactivated ␣-synuclein. Conclusion: DAT/␣-synuclein interaction at the cell surface alters the ionic coupling of DAT. Significance: This interaction modulates dopamine transmission and thus neuronal function.

“…Culture of N27 Cells, HDAC Inhibitor Treatment, and Oxidative Stress Treatment-Dopaminergic cells derived from embryonic day 12 rat mesencephalon and immortalized with the SV40 large T antigen designated 1RB3AN27, (N27 cells) were used (37). N27 cells were cultured in 6-well or 24-well plates in RPMI 1640 medium containing 10% fetal bovine serum.…”

Section: Methodsmentioning

confidence: 99%

Phenylbutyrate Up-regulates the DJ-1 Protein and Protects Neurons in Cell Culture and in Animal Models of Parkinson Disease

Zhou

Bercury

Cummiskey

et al. 2011

Self Cite

159

110

Parkinson disease is caused by the death of midbrain dopamine neurons from oxidative stress, abnormal protein aggregation, and genetic predisposition. In 2003, Bonifati et al. (23) found that a single amino acid mutation in the DJ-1 protein was associated with early-onset, autosomal recessive Parkinson disease (PARK7). The mutation L166P prevents dimerization that is essential for the antioxidant and gene regulatory activity of the DJ-1 protein. Because low levels of DJ-1 cause Parkinson, we reasoned that overexpression might stop the disease. We found that overexpression of DJ-1 improved tolerance to oxidative stress by selectively up-regulating the rate-limiting step in glutathione synthesis. When we imposed a different metabolic insult, A53T mutant ␣-synuclein, we found that DJ-1 turned on production of the chaperone protein Hsp-70 without affecting glutathione synthesis. After screening a number of small molecules, we have found that the histone deacetylase inhibitor phenylbutyrate increases DJ-1 expression by 300% in the N27 dopamine cell line and rescues cells from oxidative stress and mutant ␣-synuclein toxicity. In mice, phenylbutyrate treatment leads to a 260% increase in brain DJ-1 levels and protects dopamine neurons against 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) toxicity. In a transgenic mouse model of diffuse Lewy body disease, long-term administration of phenylbutyrate reduces ␣-synuclein aggregation in brain and prevents age-related deterioration in motor and cognitive function. We conclude that drugs that up-regulate DJ-1 gene expression may slow the progression of Parkinson disease by moderating oxidative stress and protein aggregation. Dopamine cell death in Parkinson disease (PD)3 results from both genetic and environmental factors (1-5). Six genes have been linked to PD including ␣-synuclein, Parkin, UCHL1, DJ-1, PINK1, and LRRK2 (6 -7). ␣-Synuclein mutations (A53T, A30P, and E46K) cause autosomal dominant forms of PD (8 -10). Even in sporadic cases of PD, aggregated ␣-synuclein has been found to be a major component of Lewy bodies (11-13). The toxicity of mutant forms of ␣-synuclein results from increased formation of oligomeric and fibrillar aggregates (14 -17). We and others (18 -22) have demonstrated that expression of A53T mutant ␣-synuclein results in protein aggregation and cell death in cultured dopamine neurons.Mutations in the DJ-1 gene (PARK7) lead to early-onset, autosomal recessive Parkinson disease (23-26). Ordinarily, DJ-1 protects cells by a number of mechanisms. The protein can self-oxidize by forming cysteine-sulfinic acid under oxidizing conditions, thereby shifting its pI from 6.1 to 5.8 (27-28). DJ-1 can sequester the cell death protein Daxx and prevent Daxx-induced apoptosis after oxidative stress (29). DJ-1 can stabilize Nrf2 (nuclear factor erythroid 2-related factor) by preventing association with its inhibitor protein, Keap1, thereby blocking the subsequent ubiquitination of Nrf2 (30).Previously, we have reported that overexpression of WT DJ-1 can prote...