Dopamine melanin‐loaded PC12 cells: a model for studies on pigmented neurons

Östergren, Anna; Svensson, Anne‐Lie; Lindquist, Nils Gunnar; Brittebo, Eva B.

doi:10.1111/j.1600-0749.2005.00239.x

Cited by 13 publications

(7 citation statements)

References 46 publications

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“…6, A and B). Fluorescent dyes were evident in fixed cells, probably because of cross-linking effects of fixation as demonstrated in previous studies with biogenic amines (30,36) or because the dyes were trapped in subcellular compartments. Furthermore, the dissipation of these dyes from the cellular compartment when the dye was washed from the extracellular compartment required several minutes and was stopped by fixation.…”

Section: Transportmentioning

confidence: 82%

Characterization of regulatory mechanisms and states of human organic cation transporter 2

Biermann

Lang²,

Gorboulev³

et al. 2006

American Journal of Physiology-Cell Physiology

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Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37 degrees C. After subtraction of unspecific uptake determined in WT at 37 degrees C or in hOCT2 at 8 degrees C saturable specific uptake of both substrates was measured. Km values of hOCT2-mediated uptake of 95 microM amiloride and 24 microM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC50 (in microM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca2+/CaM complex (-55 +/- 5%, n = 10 and -63 +/- 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (-54 +/- 5%, n = 14, and -31 +/- 6%, n = 26) and PKA (-16 +/- 5%, n = 16, and -18 +/- 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 +/- 6%, n = 8, and +55 +/- 17%, n = 16). Inhibition of Ca2+/CaM complex resulted in a significant decrease of Vmax (160-99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (-22 +/- 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca2+/CaM complex causes changes in transport capacity via hOCT2 trafficking.

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Section: Transportmentioning

confidence: 82%

Characterization of regulatory mechanisms and states of human organic cation transporter 2

Biermann

Lang²,

Gorboulev³

et al. 2006

American Journal of Physiology-Cell Physiology

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“…PC12 cells [51] (obtained from A–L Svensson, Uppsala university [52]) were plated at a density of 10 000 cells/well in a 96 well plate (cell+, Sarstedts, Sweden) and incubated for 18 h at 37°C in RPMI 1640 medium (Invitrogen, Stockholm, Sweden) supplemented with 10% dialyzed FBS (fetal bovine serum, Invitrogen). Aβ concentration in the different fractions was determined by Aβ42 ELISA after SDS-denaturation (see above).…”

Section: Methodsmentioning

confidence: 99%

Large Aggregates Are the Major Soluble Aβ Species in AD Brain Fractionated with Density Gradient Ultracentrifugation

et al. 2012

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Soluble amyloid-β (Aβ) aggregates of various sizes, ranging from dimers to large protofibrils, have been associated with neurotoxicity and synaptic dysfunction in Alzheimer's Disease (AD). To investigate the properties of biologically relevant Aβ species, brain extracts from amyloid β protein precursor (AβPP) transgenic mice and AD patients as well as synthetic Aβ preparations were separated by size under native conditions with density gradient ultracentrifugation. The fractionated samples were then analyzed with atomic force microscopy (AFM), ELISA, and MTT cell viability assay. Based on AFM appearance and immunoreactivity to our protofibril selective antibody mAb158, synthetic Aβ42 was divided in four fractions, with large aggregates in fraction 1 and the smallest species in fraction 4. Synthetic Aβ aggregates from fractions 2 and 3 proved to be most toxic in an MTT assay. In AβPP transgenic mouse brain, the most abundant soluble Aβ species were found in fraction 2 and consisted mainly of Aβ40. Also in AD brains, Aβ was mainly found in fraction 2 but primarily as Aβ42. All biologically derived Aβ from fraction 2 was immunologically discriminated from smaller species with mAb158. Thus, the predominant species of biologically derived soluble Aβ, natively separated by density gradient ultracentrifugation, were found to match the size of the neurotoxic, 80–500 kDa synthetic Aβ protofibrils and were equally detected with mAb158.

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“…Synthetic dopamine melanin (DAM), a widely used model of human NM (see for example, [41]) was produced by incubation for 2 weeks of 1 mM dopamine hydrochloride with 0.1 mM cupric sulfate pentahydrate in phosphate buffered saline (pH 7.4). The resulting solid oxidation product was then washed in distilled water and centrifuged four times before being lyophilized.…”

Section: Methodsmentioning

confidence: 99%

Neuromelanin is an immune stimulator for dendritic cells in vitro

et al. 2011

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BackgroundParkinson's disease (PD) is characterized at the cellular level by a destruction of neuromelanin (NM)-containing dopaminergic cells and a profound reduction in striatal dopamine. It has been shown recently that anti-melanin antibodies are increased in sera of Parkinson patients, suggesting that NM may act as an autoantigen. In this study we tested whether NM is being recognized by dendritic cells (DCs), the major cell type for inducing T- and B-cell responses in vivo. This recognition of NM by DCs is a prerequisite to trigger an adaptive autoimmune response directed against NM-associated structures.ResultsMurine DCs were treated with NM of substantia nigra (SN) from human subjects or with synthetic dopamine melanin (DAM). DCs effectively phagocytized NM and subsequently developed a mature phenotype (CD86high/MHCIIhigh). NM-activated DCs secreted the proinflammatory cytokines IL-6 and TNF-α. In addition, they potently triggered T cell proliferation in a mixed lymphocyte reaction, showing that DC activation was functional to induce a primary T cell response. In contrast, DAM, which lacks the protein and lipid components of NM but mimics the dopamine-melanin backbone of NM, had only very little effect on DC phenotype and function.ConclusionsNM is recognized by DCs in vitro and triggers their maturation. If operative in vivo, this would allow the DC-mediated transport and presentation of SN antigens to the adaptive immune system, leading to autoimmmunity in susceptible individuals. Our data provide a rationale for an autoimmune-based pathomechanism of PD with NM as the initial trigger.

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Dopamine melanin‐loaded PC12 cells: a model for studies on pigmented neurons

Cited by 13 publications

References 46 publications

Characterization of regulatory mechanisms and states of human organic cation transporter 2

Characterization of regulatory mechanisms and states of human organic cation transporter 2

Large Aggregates Are the Major Soluble Aβ Species in AD Brain Fractionated with Density Gradient Ultracentrifugation

Neuromelanin is an immune stimulator for dendritic cells in vitro

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