Lethal Factor Domain-Mediated Delivery of Nurr1 Transcription Factor Enhances Tyrosine Hydroxylase Activity and Protects from Neurotoxin-Induced Degeneration of Dopaminergic Cells

Abstract

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.

Neonatal Nicotine Exposure Primes Midbrain Neurons to a Dopaminergic Phenotype and Increases Adult Drug Consumption

“…Consistent with this, recent studies have shown that the number of neurons expressing tyrosine hydroxylase (TH), the DAsynthesizing enzyme, increases in the adult mouse substantia nigra compacta in response to photoperiod (28) and in the VTA following deep brain stimulation (29), social defeat (30), and chronic exposure to methamphetamine (31). Given that the transcription factor Nurr1 (32) is involved in the acquisition (33) and maintenance (34) of the DAergic phenotype (35), and that its expression is activity dependent (36,37), we investigated whether Nurr1 and TH expression are affected by nicotine-mediated circuit activation during development.…”

Novel Tools towards Magnetic Guidance of Neurite Growth: (I) Guidance of Magnetic Nanoparticles into Neurite Extensions of Induced Human Neurons and In Vitro Functionalization with RAS Regulating Proteins

“…Although various differentiation protocols were described for generating mDA neurons, we believe that only viral transduction-free and cDNA transfection-free approaches are worthy of consideration for cells in replacement therapies. Besides the small molecules and the cytokines used in this study, cell penetrating peptides of recombinant transcription factors [56,57] or in situ transdifferentiation of astrocytes [58,59,60] avoid negative side effects, consequently being eligible for PD therapies.…”

Regulation of Social Stress and Neural Degeneration by Activity-Regulated Genes and Epigenetic Mechanisms in Dopaminergic Neurons

“…A second strong behavioral response was found upon downregulation of the fly ARG-TF Hr38, a homolog of the human Nurr1/NR4A2 ARG, whose continued transcription is required for maintenance of adult mammalian dopaminergic neurons (DANs) [33]. Moreover, increasing Nurr1 expression helps generate DANs from iPSCs [34], while the expression of a nuclear-targeted Nurr1 fragment in dopaminergic SH-SY5Y cells increased tyrosine hydroxylase expression and protected cells from the DAN-specific neurotoxin 6-hydroxydopamine [35]. Nurr1 expression is reduced in some cases of PD, and the authors speculate that protein expression of fragments of Nurr1 may alleviate PD neurodegeneration [35].…”

“…Moreover, increasing Nurr1 expression helps generate DANs from iPSCs [34], while the expression of a nuclear-targeted Nurr1 fragment in dopaminergic SH-SY5Y cells increased tyrosine hydroxylase expression and protected cells from the DAN-specific neurotoxin 6-hydroxydopamine [35]. Nurr1 expression is reduced in some cases of PD, and the authors speculate that protein expression of fragments of Nurr1 may alleviate PD neurodegeneration [35]. Furthermore, HDAC inhibitors increase dopamine aminotransferase (DAT) expression in DANs via acetylation of Nurr1 promoters and increased Nurr1 transcription [59].…”