The role of developmental transcription factors in maintenance of neuronal properties and in disease remains poorly understood. Lmx1a and Lmx1b are key transcription factors required for the early specification of ventral midbrain dopamine (mDA) neurons. Here we show that conditional ablation of Lmx1a and Lmx1b after mDA neuron specification resulted in abnormalities that show striking resemblance to early cellular abnormalities seen in Parkinson's disease. We found that Lmx1b was required for the normal execution of the autophagic-lysosomal pathway and for the integrity of dopaminergic nerve terminals and long-term mDA neuronal survival. Notably, human LMX1B expression was decreased in mDA neurons in brain tissue affected by Parkinson's disease. Thus, these results reveal a sustained and essential requirement of Lmx1b for the function of midbrain mDA neurons and suggest that its dysfunction is associated with Parkinson's disease pathogenesis.

␣-synuclein, a protein enriched in Lewy bodies and highly implicated in neurotoxicity in Parkinson's disease, is distributed both at nerve terminals and in the cell nucleus. Here we show that a nuclear derivative of ␣-synuclein induces more pronounced changes at the gene expression level in mouse primary dopamine (DA) neurons compared to a derivative that is excluded from the nucleus. Moreover, by RNA sequencing we analyzed the extent of genome-wide effects on gene expression resulting from expression of human ␣-synuclein in primary mouse DA neurons. The results implicated the transcription factor Nurr1 as a key dysregulated target of ␣-synuclein toxicity. Forced Nurr1 expression restored the expression of hundreds of dysregulated genes in primary DA neurons expressing ␣-synuclein, and therefore prompted us to test the possibility that Nurr1 can be pharmacologically targeted by bexarotene, a ligand for the retinoid X receptor that forms heterodimers with Nurr1. Although our data demonstrated that bexarotene was ineffective in neuroprotection in rats in vivo, the results revealed that bexarotene has the capacity to coregulate subsets of Nurr1 target genes including the receptor tyrosine kinase subunit Ret. Moreover, bexarotene was able to restore dysfunctional Ret-dependent neurotrophic signaling in ␣-synucleinoverexpressing mouse DA neurons. These data highlight the role of the Nurr1-Ret signaling pathway as a target of ␣-synuclein toxicity and suggest that retinoid X receptor ligands with appropriate pharmacological properties could have therapeutic potential in Parkinson's disease.

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Exogenous cell replacement represents a potent treatment option for Parkinson's disease. However, the low survival rate of transplanted dopaminergic neurons (DA) calls for methodological improvements. Here we evaluated a method to combine transient genetic modification of neuronal progenitor cells with an optimized cell culture protocol prior to intrastriatal transplantation into 6-hydroxydopamine (6-OHDA) unilateral lesioned rats. Plasmid-based delivery of brain-derived neurotrophic factor (BDNF) increases the number of DA neurons, identified by tyrosine hydroxylase immunoreactivity (TH-ir), by 25% in vitro, compared to enhanced green fluorescence protein (EGFP)-transfected controls. However, the nucleofection itself, especially the cell detachment and reseeding procedure, decreases the TH-ir neuron number to 40% compared with nontransfected control cultures. To circumvent this drawback we established the colayer method, which contains a mix of nucleofected cells reseeded on top of an adherent sister culture in a ratio 1:3. In this setup TH-ir neuron number remains high and could be further increased by 25% after BDNF transfection. Comparison of both cell culture procedures (standard and colayer) after intrastriatal transplantation revealed a similar DA neuron survival as seen in vitro. Two weeks after grafting TH-ir neuron number was strongly reduced in animals receiving the standard EGFP-transfected cells (271 ± 62) compared to 1,723 ± 199 TH-ir neurons in the colayer group. In contrast to the in vitro results, no differences in the number of grafted TH-ir neurons were observed between BDNF, EGFP, and nontransfected colayer groups, neither 2 nor 13 weeks after transplantation. Likewise, amphetamine and apomorphine-induced rotational behavior improved similarly over time in all groups. Nevertheless, the colayer protocol provides an efficient way for neurotrophic factor release by transplanted progenitor cells and will help to study the effects of candidate factors on survival and integration of transplanted DA neurons.

Neuronal progenitor cells (NPCs) possess high potential for use in regenerative medicine. To overcome their limited mitotic competence, various immortalization strategies have been applied that allow their prolonged maintenance and expansion in vitro. Such immortalized cells can be used for the design and discovery of new cell-based therapies for neurodegenerative diseases, such as Parkinson's disease. We immortalized rat ventral mesencephalic NPCs by using SV40 large T antigen (SV40Tag). All cell clones displayed a two- to three-fold higher proliferation rate compared with the primary cells. In order to induce dopaminergic differentiation of generated cell clones, both glial-derived neurotrophic factor and di-butyryl cyclic adenosine monophosphate were applied. Treated cells were then characterized regarding the expression of dopaminergic lineage markers, differentiation of various cell populations, calcium imaging in the presence of kainate, and immunohistochemistry after intrastriatal transplantation. Treated cells displayed morphological maturation, and calcium imaging revealed neuronal properties in the presence of kainate. These cells also expressed low mRNA levels of the dopamine transporter and tyrosine hydroxylase (TH), although no TH-immunopositive neurons were found. Intrastriatal transplantation into the neurotoxin-lesioned rats did not induce further differentiation. As an alternative approach, we silenced SV40Tag with short interfering RNA, but this was not sufficient to trigger differentiation into dopaminergic neurons. Nevertheless, neuronal and glial cells were detected as shown by beta-tubulin type III and glial fibrillary acidic protein staining, respectively. SV40Tag cells are suitable for carrying out controlled genetic modifications as shown by overexpression of enhanced green fluorescence protein after efficient non-viral transfection.

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The original version of this article unfortunately contained a mistake. The spelling of Daniela Ragancokova's surname is incorrect. The correct surname is given above.The online version of the original article can be found at http://dx.doi.