Standard cell culture systems impose environmental oxygen (O 2 ) levels of 20%, whereas actual tissue O 2 levels in both developing and adult brain are an order of magnitude lower. To address whether proliferation and differentiation of CNS precursors in vitro are influenced by the O 2 environment, we analyzed embryonic day 12 rat mesencephalic precursor cells in traditional cultures with 20% O 2 and in lowered O 2 (3 Ϯ 2%). Proliferation was promoted and apoptosis was reduced when cells were grown in lowered O 2 , yielding greater numbers of precursors. The differentiation of precursor cells into neurons with specific neurotransmitter phenotypes was also significantly altered. The percentage of neurons of dopaminergic phenotype increased to 56% in lowered O 2 compared with 18% in 20% O 2 . Together, the increases in total cell number and percentage of dopaminergic neurons resulted in a ninefold net increase in dopamine neuron yield. Differential gene expression analysis revealed more abundant messages for FGF8, engrailed-1, and erythropoietin in lowered O 2 . Erythropoietin supplementation of 20% O 2 cultures partially mimicked increased dopaminergic differentiation characteristic of CNS precursors cultured in lowered O 2 . These data demonstrate increased proliferation, reduced cell death, and enhanced dopamine neuron generation in lowered O 2 , making this method an important advance in the ex vivo generation of specific neurons for brain repair. Key words: CNS precursors; CNS stem cells; dopaminergic neurons; erythropoietin; oxygen; Parkinson's diseaseCultured CNS stem cells have proved useful in defining the pathways that lead to generation of neurons and glia (McKay, 1997). These cells self-renew, and after mitogen withdrawal, differentiate into neurons, astrocytes and oligodendrocytes in predictable proportions (Johe et al., 1996;McKay, 1997). Single extrinsic factors can shift the fate of CNS stem cells toward specific cell lineages (Johe et al., 1996;Panchision et al., 1998). The potential therapeutic application of CNS stem cells in common degenerative and ischemic diseases has become a major focus of research. The generation of dopaminergic neurons from CNS precursors is of special interest given the promising results of fetal cell transplantation in patients with Parkinson's disease (Olanow et al., 1996; Piccini at al., 1999;Freeman et al., 2000).In clinical settings, gases are appreciated as primary variables in organ survival, with O 2 as the critical gas parameter. However, traditional CNS stem cell culture (as well as virtually all other ex vivo cell culture) is performed in nonphysiologically high O 2 . Standard tissue culture incubator conditions are 5% CO 2 and 95% air, which exposes cells to a 20% O 2 environment. In mammalian brain, interstitial tissue O 2 levels range from ϳ1 to 5% (Table 1). We tested the effects of culturing CNS progenitor cells in physiological "lowered" (3 Ϯ 2%) O 2 , comparing the cultures with those grown in the usual 20% O 2 . Our results indicate that oxygen lowere...

Dopaminergic transmission within limbic regions of the brain is highly dependent on the regulation of D2 receptor activity. Here we show that the neuronal calcium sensor-1 (NCS-1) can mediate desensitization of D2 dopamine receptors. Analysis of D2 receptors expressed in human embryonic kidney 293 cells indicates that NCS-1 attenuates agonist-induced receptor internalization via a mechanism that involves a reduction in D2 receptor phosphorylation. This effect of NCS-1 was accompanied by an increase in D2 receptor-mediated cAMP inhibition after dopamine stimulation. The ability of NCS-1 to modulate D2 receptor signaling was abolished after a single amino acid mutation in NCS-1 that has been shown to impair the calcium-binding properties of NCS-1. Coimmunoprecipitation experiments from striatal neurons reveal that NCS-1 is found in association with both the D2 receptor and G-protein-coupled receptor kinase 2, a regulator of D2 receptor desensitization. Colocalization of NCS-1 and D2 receptors was examined in both primate and rodent brain. In striatum, NCS-1 and D2 receptors were found to colocalize within sites of synaptic transmission and in close proximity to intracellular calcium stores. NCS-1-D2 receptor interaction may serve to couple dopamine and calcium signaling pathways, thereby providing a critical component in the regulation of dopaminergic signaling in normal and diseased brain.

We have used a yeast two-hybrid approach to uncover protein interactions involving the D2-like subfamily of dopamine receptors. Using the third intracellular loop of the D2S and D3 dopamine receptors as bait to screen a human brain cDNA library, we identified filamin A (FLN-A) as a protein that interacts with both the D2 and D3 subtypes. The interaction with FLN-A was specific for the D2 and D3 receptors and was independently confirmed in pulldown and coimmunoprecipitation experiments. Deletion mapping localized the dopamine receptor-FLN-A interaction to the N-terminal segment of the D2 and D3 dopamine receptors and to repeat 19 of FLN-A. In cultures of dissociated rat striatum, FLN-A and D2 receptors colocalized throughout neuronal somata and processes as well as in astrocytes. Expression of D2 dopamine receptors in FLN-A-deficient M2 melanoma cells resulted in predominant intracellular localization of the D2 receptors, whereas in FLN-A-reconstituted cells, the D2 receptor was predominantly localized at the plasma membrane. These results suggest that FLN-A may be required for proper cell surface expression of the D2 dopamine receptors. Association of D2 and D3 dopamine receptors with FLN-A provides a mechanism whereby specific dopamine receptor subtypes may be functionally linked to downstream signaling components via the actin cytoskeleton. I mbalances in dopaminergic signaling are implicated in many neuropsychiatric and motor disorders, including schizophrenia and Parkinson's disease (1). In mammalian brain, dopaminergic signaling is mediated via a cohort of dopamine receptors. Among the cloned dopamine receptor subtypes, the D2-like receptors (D2, D3, and D4) are the major target of antipsychotics, both typical and atypical, as well as anti-Parkinson's drugs (1). These receptors mediate intracellular signaling cascades by coupling to inhibitory subsets of heterotrimeric GTP-binding (G) proteins. In a variety of cell types, D2-like receptor signaling modulates calcium, potassium, and sodium currents through specific regulation of ion channel activities (2, 3). The activation of D2-like receptors also has been implicated in the regulation of cellular morphogenesis (4) and in the maintenance of neuronal structure in adult brain (5-7). Although the D2-like receptors appear to activate discrete signal transduction pathways, the question of whether individual D2-like receptors subserve distinct functional roles is an issue that has not yet been satisfactorily addressed.To better understand the regulation of dopamine receptor signaling, we are interested in identifying dopamine receptor-interacting proteins. Identification of dopamine receptor-interacting proteins unique to specific receptor subtypes may provide important clues to how functional differences between dopamine receptor subtypes are manifested. We conducted yeast two-hybrid screens of a human brain cDNA library, with the third intracellular loop of the D2S and D3 receptors as bait. Using this approach, we identified filamin A (FLN-A) as a dopamine rece...