The orphan transcription factor nuclear receptor-related 1 protein (Nurr1, also known as NR4A2) plays a key role in embryonic development and maintenance of mesencephalic dopaminergic neurons in the substantia nigra. Nurr1 deficiency is associated with Parkinson's disease where dopaminergic neurons degenerate suggesting that counter-regulation of Nurr1 activity may have therapeutic effects. Here, we bacterially expressed and isolated a human Nurr1 fusion protein containing a N-terminal cell delivery domain derived from detoxified anthrax lethal factor followed by wild type ubiquitin with deubiquitinating enzyme recognition site for intracellular cleavage. Addition of the Nurr1 fusion protein to dopaminergic SH-SY5Y cells generated a cleaved, cytosolic Nurr1-containing fragment which was associated with increased levels of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Promoter-activity assays confirmed that exposure of cells to full-length Nurr1 fusion protein activated not only its cognate human tyrosine hydroxylase promoter but also the corresponding mouse sequence, although at a reduced efficiency. Using 6-hydroxydopamine as a dopaminergic cell specific neurotoxin, we demonstrate that full-length Nurr1 fusion protein promotes a concentration-dependent protection from this toxic insult. Altogether, the enhancement of tyrosine hydroxylase in naïve dopaminergic cells and the protective effects in a cellular model of Parkinson's disease suggest that full-length Nurr1 fusion protein may contribute to the development of a novel concept of protein-based therapy.

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SUMMARYThe virulence of Bacillus anthracis is linked to the secretion of anthrax lethal toxin and anthrax edema toxin. These binary toxins consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds either of two specific cell surface receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding, endocytosis, and cytoplasmic translocation of LF and EF. The cellular distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals is not fully elucidated. Herein, we describe a novel assay to image anthrax toxin intoxication in live animals, and we use the assay to visualize TEM-8- and CMG-2-dependent intoxication. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were co-administered to transgenic mice that ubiquitously express a red fluorescent protein in the absence of Cre activity and a green fluorescent protein in the presence of Cre activity, anthrax toxin intoxication could be visualized at single-cell resolution by confocal microscopy. By using this assay, we show that CMG-2 is critical for intoxication in the liver and heart, whereas TEM-8 is required for full intoxication in the kidney and spleen. Other tissues examined were largely unaffected by single deficiences in either receptor, suggesting extensive overlap in TEM-8 and CMG-2 expression. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for identifying on- and off-targets for reengineered toxin variants in the clinical development of cancer treatments.BackgroundAssays for imaging of anthrax toxin intoxication in animals are not available.ResultsAnthrax toxin-Cre fusions combined with fluorescent Cre reporter mice enabled imaging of anthrax toxin intoxication in animals.ConclusionShared and distinct functions of toxin receptors in cellular entry were uncovered. Significance. A simple and versatile assay for anthrax toxin intoxication is described.

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