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Rgs6 is required for adult maintenance of dopaminergic neurons in the ventral substantia nigra.

Bifsha P, Yang J, Fisher RA, Drouin J - PLoS Genet. (2014)

Bottom Line: Unilateral cell loss is accompanied by contralateral degenerating neurons that exhibit smaller cell size, altered morphology and reduced dendritic network.The degenerating neurons have low levels of tyrosine hydroxylase (TH) and decreased nuclear Pitx3; accordingly, expression of many Pitx3 target gene products is altered, including Vmat2, Bdnf, Aldh1a1 (Adh2) and Fgf10.Rgs6-dependent protection is thus critical for adult survival and maintenance of the vSNc mDA neurons that are most affected in PD.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Génétique Moléculaire, Institut de Recherches Cliniques de Montréal (IRCM) Montréal, Quebec, Canada; Division of Experimental Medicine, McGill University, Montréal, Quebec, Canada.

ABSTRACT
Parkinson disease (PD) is characterized by the preferential, but poorly understood, vulnerability to degeneration of midbrain dopaminergic (mDA) neurons in the ventral substantia nigra compacta (vSNc). These sensitive mDA neurons express Pitx3, a transcription factor that is critical for their survival during development. We used this dependence to identify, by flow cytometry and expression profiling, the negative regulator of G-protein signaling Rgs6 for its restricted expression in these neurons. In contrast to Pitx3-/- mDA neurons that die during fetal (vSNc) or post-natal (VTA) period, the vSNc mDA neurons of Rgs6-/- mutant mice begin to exhibit unilateral signs of degeneration at around 6 months of age, and by one year cell loss is observed in a fraction of mice. Unilateral cell loss is accompanied by contralateral degenerating neurons that exhibit smaller cell size, altered morphology and reduced dendritic network. The degenerating neurons have low levels of tyrosine hydroxylase (TH) and decreased nuclear Pitx3; accordingly, expression of many Pitx3 target gene products is altered, including Vmat2, Bdnf, Aldh1a1 (Adh2) and Fgf10. These low TH neurons also express markers of increased dopamine signaling, namely increased DAT and phospho-Erk1/2 expression. The late onset degeneration may reflect the protective action of Rgs6 against excessive DA signaling throughout life. Rgs6-dependent protection is thus critical for adult survival and maintenance of the vSNc mDA neurons that are most affected in PD.

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Subset-specific expression signatures of mDA neurons.(A) Differentially expressed probesets were clustered in an unbiased manner into three-dimensional Cartesian-type coordinates defined according to the relative enrichment attributes for each probeset in the comparisons between SNc WT/VTA WT, SNc KO/SNc WT and VTA KO/VTA WT. Each cluster was named according to the observed preferential expression pattern (label and shown in red in diagrams). The heatmaps show relative enrichments for 20 genes that are highly enriched and/or that have documented functions (the complete gene lists for each subset is provided in Table S1). The total number of genes (not probesets) in each cluster is indicated together with the number of those that are either activated or repressed by Pitx3 based on the VTA KO/WT comparison. (B) qRT-PCR quantification of mRNAs with preferential expression in either SN or VTA assessed in FACS-sorted cells from WT or Pitx3−/− (KO) tissues. mRNA levels are normalized relative to Gapdh mRNA. Data are shown as means ± S.D.
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pgen-1004863-g002: Subset-specific expression signatures of mDA neurons.(A) Differentially expressed probesets were clustered in an unbiased manner into three-dimensional Cartesian-type coordinates defined according to the relative enrichment attributes for each probeset in the comparisons between SNc WT/VTA WT, SNc KO/SNc WT and VTA KO/VTA WT. Each cluster was named according to the observed preferential expression pattern (label and shown in red in diagrams). The heatmaps show relative enrichments for 20 genes that are highly enriched and/or that have documented functions (the complete gene lists for each subset is provided in Table S1). The total number of genes (not probesets) in each cluster is indicated together with the number of those that are either activated or repressed by Pitx3 based on the VTA KO/WT comparison. (B) qRT-PCR quantification of mRNAs with preferential expression in either SN or VTA assessed in FACS-sorted cells from WT or Pitx3−/− (KO) tissues. mRNA levels are normalized relative to Gapdh mRNA. Data are shown as means ± S.D.

Mentions: In order to identify genes responsible for the differential vulnerability of vSNc mDA neurons, we devised a strategy to isolate FACS-purified Pitx3-dependent and Pitx3-independent mDA neurons and compare their transcriptomes (Fig. 1). By birth, the SNc of Pitx3−/− pups is completely depleted of Pitx3-positive neurons but the dorsal Pitx3-negative neurons are spared [12]. After dissection of SN and VTA from midbrain slices of mice expressing TH-EGFP, FACS-sorting of TH-EGFP-positive neurons yielded a pure dSNc Pitx3-negative population from Pitx3−/− brains and mixed (∼80% Pitx3-positive and ∼20% Pitx3-negative) SN mDA populations from wild-type animals. The comparison of their transcriptomes defined vSNc- and dSNc-enriched genes (Fig. 2A). In VTA, Pitx3 deficiency leaves the 50% Pitx3-expressing mDA neurons intact at birth but they die within the next three months [12]. Comparison of VTA TH-EGFP cell expression profiles from Pitx3−/− and WT mice will thus identify genes which have Pitx3-dependent expression. RNA extracted from FACS-sorted cells (Fig. 1) was used to generate probes for hybridization in duplicates to Affymetrix Mouse Gene 1.0ST microarrays and determination of expression profiles. Unbiased clustering of the 1813 differentially expressed genes (fold changes >1.5, p≤0.05 and signal ≥60) into seven clusters defined genes that are expressed in specific subsets of mDA neurons and/or that are Pitx3-dependent in VTA (Fig. 2A). qRT-PCR analyses confirmed the expected enrichment for vSNc (Girk2, DAT), dSNc (Calb1) and VTA (Otx2, Calb1) markers (Fig. 2B). Further, many genes previously characterized for their subset-specific expression (marked by stars in Fig. 2A) validate the profiling data; these include Calb1/2 for dSNc, Otx2 for VTA, Kcnj6 (Girk2) for vSNc, Lpl for VTA, Aldh1a1 for vSNc, Slc6a3 (DAT) for vSNc, Lix1 for SNc [20], [30]–[32]. The complete list of genes in each cluster is provided in Table S1.


Rgs6 is required for adult maintenance of dopaminergic neurons in the ventral substantia nigra.

Bifsha P, Yang J, Fisher RA, Drouin J - PLoS Genet. (2014)

Subset-specific expression signatures of mDA neurons.(A) Differentially expressed probesets were clustered in an unbiased manner into three-dimensional Cartesian-type coordinates defined according to the relative enrichment attributes for each probeset in the comparisons between SNc WT/VTA WT, SNc KO/SNc WT and VTA KO/VTA WT. Each cluster was named according to the observed preferential expression pattern (label and shown in red in diagrams). The heatmaps show relative enrichments for 20 genes that are highly enriched and/or that have documented functions (the complete gene lists for each subset is provided in Table S1). The total number of genes (not probesets) in each cluster is indicated together with the number of those that are either activated or repressed by Pitx3 based on the VTA KO/WT comparison. (B) qRT-PCR quantification of mRNAs with preferential expression in either SN or VTA assessed in FACS-sorted cells from WT or Pitx3−/− (KO) tissues. mRNA levels are normalized relative to Gapdh mRNA. Data are shown as means ± S.D.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263397&req=5

pgen-1004863-g002: Subset-specific expression signatures of mDA neurons.(A) Differentially expressed probesets were clustered in an unbiased manner into three-dimensional Cartesian-type coordinates defined according to the relative enrichment attributes for each probeset in the comparisons between SNc WT/VTA WT, SNc KO/SNc WT and VTA KO/VTA WT. Each cluster was named according to the observed preferential expression pattern (label and shown in red in diagrams). The heatmaps show relative enrichments for 20 genes that are highly enriched and/or that have documented functions (the complete gene lists for each subset is provided in Table S1). The total number of genes (not probesets) in each cluster is indicated together with the number of those that are either activated or repressed by Pitx3 based on the VTA KO/WT comparison. (B) qRT-PCR quantification of mRNAs with preferential expression in either SN or VTA assessed in FACS-sorted cells from WT or Pitx3−/− (KO) tissues. mRNA levels are normalized relative to Gapdh mRNA. Data are shown as means ± S.D.
Mentions: In order to identify genes responsible for the differential vulnerability of vSNc mDA neurons, we devised a strategy to isolate FACS-purified Pitx3-dependent and Pitx3-independent mDA neurons and compare their transcriptomes (Fig. 1). By birth, the SNc of Pitx3−/− pups is completely depleted of Pitx3-positive neurons but the dorsal Pitx3-negative neurons are spared [12]. After dissection of SN and VTA from midbrain slices of mice expressing TH-EGFP, FACS-sorting of TH-EGFP-positive neurons yielded a pure dSNc Pitx3-negative population from Pitx3−/− brains and mixed (∼80% Pitx3-positive and ∼20% Pitx3-negative) SN mDA populations from wild-type animals. The comparison of their transcriptomes defined vSNc- and dSNc-enriched genes (Fig. 2A). In VTA, Pitx3 deficiency leaves the 50% Pitx3-expressing mDA neurons intact at birth but they die within the next three months [12]. Comparison of VTA TH-EGFP cell expression profiles from Pitx3−/− and WT mice will thus identify genes which have Pitx3-dependent expression. RNA extracted from FACS-sorted cells (Fig. 1) was used to generate probes for hybridization in duplicates to Affymetrix Mouse Gene 1.0ST microarrays and determination of expression profiles. Unbiased clustering of the 1813 differentially expressed genes (fold changes >1.5, p≤0.05 and signal ≥60) into seven clusters defined genes that are expressed in specific subsets of mDA neurons and/or that are Pitx3-dependent in VTA (Fig. 2A). qRT-PCR analyses confirmed the expected enrichment for vSNc (Girk2, DAT), dSNc (Calb1) and VTA (Otx2, Calb1) markers (Fig. 2B). Further, many genes previously characterized for their subset-specific expression (marked by stars in Fig. 2A) validate the profiling data; these include Calb1/2 for dSNc, Otx2 for VTA, Kcnj6 (Girk2) for vSNc, Lpl for VTA, Aldh1a1 for vSNc, Slc6a3 (DAT) for vSNc, Lix1 for SNc [20], [30]–[32]. The complete list of genes in each cluster is provided in Table S1.

Bottom Line: Unilateral cell loss is accompanied by contralateral degenerating neurons that exhibit smaller cell size, altered morphology and reduced dendritic network.The degenerating neurons have low levels of tyrosine hydroxylase (TH) and decreased nuclear Pitx3; accordingly, expression of many Pitx3 target gene products is altered, including Vmat2, Bdnf, Aldh1a1 (Adh2) and Fgf10.Rgs6-dependent protection is thus critical for adult survival and maintenance of the vSNc mDA neurons that are most affected in PD.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Génétique Moléculaire, Institut de Recherches Cliniques de Montréal (IRCM) Montréal, Quebec, Canada; Division of Experimental Medicine, McGill University, Montréal, Quebec, Canada.

ABSTRACT
Parkinson disease (PD) is characterized by the preferential, but poorly understood, vulnerability to degeneration of midbrain dopaminergic (mDA) neurons in the ventral substantia nigra compacta (vSNc). These sensitive mDA neurons express Pitx3, a transcription factor that is critical for their survival during development. We used this dependence to identify, by flow cytometry and expression profiling, the negative regulator of G-protein signaling Rgs6 for its restricted expression in these neurons. In contrast to Pitx3-/- mDA neurons that die during fetal (vSNc) or post-natal (VTA) period, the vSNc mDA neurons of Rgs6-/- mutant mice begin to exhibit unilateral signs of degeneration at around 6 months of age, and by one year cell loss is observed in a fraction of mice. Unilateral cell loss is accompanied by contralateral degenerating neurons that exhibit smaller cell size, altered morphology and reduced dendritic network. The degenerating neurons have low levels of tyrosine hydroxylase (TH) and decreased nuclear Pitx3; accordingly, expression of many Pitx3 target gene products is altered, including Vmat2, Bdnf, Aldh1a1 (Adh2) and Fgf10. These low TH neurons also express markers of increased dopamine signaling, namely increased DAT and phospho-Erk1/2 expression. The late onset degeneration may reflect the protective action of Rgs6 against excessive DA signaling throughout life. Rgs6-dependent protection is thus critical for adult survival and maintenance of the vSNc mDA neurons that are most affected in PD.

Show MeSH
Related in: MedlinePlus