Lmx1a-Dependent Activation of miR-204/211 Controls the Timing of Nurr1-Mediated Dopaminergic Differentiation

Abstract

The development of midbrain dopaminergic (DA) neurons requires a fine temporal and spatial regulation of a very specific gene expression program. Here, we report that during mouse brain development, the microRNA (miR-) 204/211 is present at a high level in a subset of DA precursors expressing the transcription factor Lmx1a, an early determinant for DA-commitment, but not in more mature neurons expressing Th or Pitx3. By combining different in vitro model systems of DA differentiation, we show that the levels of Lmx1a influence the expression of miR-204/211. Using published transcriptomic data, we found a significant enrichment of miR-204/211 target genes in midbrain dopaminergic neurons where Lmx1a was selectively deleted at embryonic stages. We further demonstrated that miR-204/211 controls the timing of the DA differentiation by directly downregulating the expression of Nurr1, a late DA differentiation master gene. Thus, our data indicate the Lmx1a-miR-204/211-Nurr1 axis as a key component in the cascade of events that ultimately lead to mature midbrain dopaminergic neurons differentiation and point to miR-204/211 as the molecular switch regulating the timing of Nurr1 expression.

The Crucial Roles of Pitx3 in Midbrain Dopaminergic Neuron Development and Parkinson’s Disease-Associated Neurodegeneration

“…Recent research has demonstrated the importance of the Lmx1a-miR-204/211-Nurr1 axis in the differentiation of mDA neurons [58]. Additionally, mice that are overexpressed with mutant alpha-synuclein and simultaneously lacking one Nurr1 allele showed the enhanced age-dependent reduction of Nurr1 protein levels, a decreased number of mDA neurons, as well as increased neuroinflammation and alpha-synuclein aggregation [59].…”

miR-218 Promotes Dopaminergic Differentiation and Controls Neuron Excitability and Neurotransmitter Release through the Regulation of a Synaptic-Related Genes Network

“…The miR-218-1 À/À and miR-218-2 À/À (referred to as KO1 and KO2, respectively) were generated by using CRISPR/Cas9 dual-nickase mediated nonhomologous end joining following a previously published protocol (Ran et al, 2013). Briefly, two pairs of single guide RNA (sgRNA) targeting the miR-218-1 and the miR-218-2 genomic loci at both bottom and top strands were designed using the CRISPR double nickase design tool from the Feng Zang laboratory (https://www.zlab.bio/resources) and evaluated for their low off-target activity.…”

“…Cas9n mRNA and sgRNA templates were amplified with T7 promoter sequence-conjugated primers and transcribed in vitro with mMESSAGE mMACHINE T7 Ultra Kit (Invitrogen) and MEGAshortscript T7 Kit (Invitrogen), respectively. The in vitro RNAs were injected into pronuclear stage fertilized mouse eggs, which were then transferred into the oviduct of pseudo-pregnant female mice (C57/Bl) (Ran et al, 2013). F1-mice carrying indels were mated with WT animals and subsequently Sangersequencing was performed on homozygotes DNA.…”

“…Mouse midbrain primary cultures (mE12.5-PCs) and high content imaging Freshly prepared single-cell suspensions from mouse embryonic midbrain, referred to as mE12.5-PCs (mouse E12.5 primary cultures), were obtained following established protocols (Prochiantz et al, 1979;di Porzio et al, 1980;Pulcrano et al, 2022). In brief, the ventral midbrains from E12.5 embryos were carefully isolated and treated with 0.25% trypsin solution containing 0.01% pancreatic DNase for 5 min at 37°C.…”

“…Separation and analysis of fluorescent cells were performed as previously described (Pulcrano et al, 2022). In brief, freshly dissected ventral midbrains of TH-GFP embryos or cells grown in monolayer were dissociated into single cells with 0.25% trypsin solution in 33 mM glucose/PBS with 0.01% pancreatic DNase.…”