<i>Rgs6−/−</i> mice as a model for Parkinsonian neurodegeneration.

<p>(A) Rgs6-dependent signaling pathways in vSNc mDA neurons control Pitx3 expression, which itself controls expression of Aldh1a1, TH, Fgf10, Vmat2 and Bdnf. Together, Rgs6 and Pitx3 define a survival pathway in these neurons. The dopamine receptor Drd2 may be a target of Rgs6 action and consistent with this hypothesis, expression of the dopamine transporter DAT is up-regulated in <i>Rgs6^−/−</i> vSNc. (B) Schematic representation of major subsets of SNc mDA neurons present at different ages in midbrains of <i>Rgs6</i>^−/− mice showing progressive degeneration (small pale green) followed by loss of vSNc mDA neurons.</p

Reduced expression of Pitx3 and its target genes in degenerating neurons.

<p>(A) Double immunofluorescence staining against TH (green) and Pitx3 (red) in SNc of control and 1 y-old <i>Rgs6</i>^−/− mice that display dysmorphic mDA neurons. Arrowheads indicate Pitx3-negative neurons of dSNc. Scale bar 20 µm. (B) Co-immunofluorescence staining against TH (green), nuclear Dapi (blue) and Vmat2 (red) in SNc of <i>WT</i> and <i>Rgs6</i>^−/− mice. Scale bar 20 µm. (C) Co-immunofluorescence staining against TH (green), nuclear Dapi (blue) and Bdnf (red) in SNc and VTA of <i>WT</i> and <i>Rgs6</i>^−/− mice. Scale bar 20 µm. (D) Double immunofluorescence staining against TH (green) and Pitx3, Aldh1a1, Fgf10 (red) in control and 1 y-old <i>Rgs6</i>^−/− mice that display dysmorphic mDA neurons. Arrowheads indicate unaffected dSNc neurons and arrows point to affected vSNc neurons. Scale bar 100 µm.</p

<i>Rgs6</i> is Required for Adult Maintenance of Dopaminergic Neurons in the Ventral Substantia Nigra

<div><p>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 <i>Pitx3^−/−</i> mDA neurons that die during fetal (vSNc) or post-natal (VTA) period, the vSNc mDA neurons of <i>Rgs6</i>^−/− 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.</p></div

Unilateral loss of Pitx3-positive dopaminergic neurons in ventral SNc of <i>Rgs6</i>^−/− mice.

<p>(A) Immunoperoxidase staining for TH on representative coronal midbrain sections showing less SNc TH+ neurons on one side of <i>Rgs6</i>^−/− mice at 1 year of age compared to sib control. Sections are identified with Bregma position. Scale bar 400 µm. (B) Number of TH+ cells in SNc and VTA of TH-stained coronal sections across midbrain (every 30 µm). Cell counts are represented as means +/− S.D. (***p<0.005). (C) Nissl staining of vSNc sections contiguous to A shows fewer cell bodies and abnormal elongated neurons in <i>Rgs6</i>^−/− mice compared to control. Scale bar 100 µm. (D) Double immunofluorescence staining for TH (green) and Pitx3 (red) on sections contiguous to A showing a marked loss of TH+Pitx3+ cells in <i>Rgs6^−/−</i> mice. Scale bar 100 µm.</p

Strategy for isolation of FACS-purified Pitx3-dependent (red) and Pitx3-independent (white) mDA neurons for expression profiling analysis.

<p>Dissected SN and VTA from newborn <i>TH-EGFP</i> transgenic <i>Pitx3^+/+</i> and <i>Pitx3^−/</i>⁻ mice were used for FACS purification of catecholaminergic neurons. The EGFP⁺ cells consists in various proportions (approximate % shown) of Pitx3+ (red), Pitx3− (white) and Pitx3del (yellow) mDA neurons, depending on the region dissected and mDA neuronal loss resulting from <i>Pitx3</i> inactivation. RNA from four cell preparations (SNc WT, VTA WT, SNc KO, VTA KO) were analyzed by hybridization in duplicates to Affymetrix Mouse Gene 1.0ST microarrays.</p

Unilateral degeneration of vSNc neurons in a subset of <i>Rgs6−/−</i> mice.

<p>(A) Immunoperoxidase staining for TH on representative coronal midbrain sections showing dysmorphic TH+ neurons (low TH staining, TH^low, inset) in ventral SNc of <i>Rgs6</i>^−/− mice. The dSNc and VTA mDA neurons are unaffected (strong/normal TH staining, inset). Scale bar 200 µm. (B) Triple staining for TH (red), Dapi (blue) and Fluoro-Jade C (FJC, green) showing presence of degenerating TH^low cells in vSNc of 1 y-old <i>Rgs6</i>^−/− mice (middle panels) and not in control vSNc (upper panels) or in dorsal SNc (lower panels). Scale bar 20 µm. (C) Bilateral cell counts of FJC⁺ mDA neurons in SNc and VTA of coronal sections from control and 1 y-old <i>Rgs6</i>^−/− mice. (D) Co-immunostaining for TH (green) and LC3B (red) in vSNc of <i>WT</i> and <i>Rgs6</i>^−/− mice. Scale bar 20 µm. Arrowheads indicate unaffected neurons while arrows point to TH^low cells. (E) Co-immunostaining for TH (green) and phosphorylated p27^Kip1 (phospho-p27, red) in vSNc of <i>WT</i> and <i>Rgs6</i>^−/− mice. Scale bar 20 µm.</p

Expression of familial PD genes is altered in degenerating neurons of vSNc.

<p>Double immunofluorescence staining against TH (green) and (A) DJ-1 (red) or (B) Pink1 (red) or (C) Lrrk2 (red) in SNc of control and 1 y-old <i>Rgs6</i>^−/− mice that display dysmorphic TH^low mDA neurons. Arrowheads indicate unaffected neurons while arrows point to TH^low cells. Scale bar 20 µm.</p

Restricted expression of Rgs6 in Pitx3-positive (Pitx3+) dopaminergic neurons of ventral SNc.

<p>(A) Rgs6 expression revealed by immunohistofluorescence (red) together with TH (green) in mDA neurons of SNc but not VTA. Scale bar 410 µm. (B) Co-immunofluorescence analysis of TH (green) and Rgs6 or Pitx3 (red, as indicated) in SNc and VTA of adult mice. Arrowheads point to Pitx3-negative or Rgs6-negative mDA neurons in dorsal SNc. Scale bar 100 µm. (C) Triple immunofluorescence staining for TH (green), Pitx3 (red) and Calb1 (blue) on tissue sections of adult mice indicates that the majority of TH+Pitx3− cells of dSNc (arrowheads) are positive for Calb1, while TH+Pitx3+ cells of vSNc are negative for Calb1, consistent with depiction in A. Scale bar 100 µm.</p

Additional file 1 of Key mRNAs and lncRNAs of pituitary that affect the reproduction of FecB + + small tail han sheep

Supplementary Material

miRNA-148a serves as a prognostic factor and suppresses migration and invasion through Wnt1 in non-small cell lung cancer

<div><p>Lung cancer is the leading cause of cancer death in the world, and aberrant expression of miRNA is a common feature during the cancer initiation and development. Our previous study showed that levels of miRNA-148a assessed by quantitative real-time polymerase chain reaction (qRT-PCR) were a good prognosis factor for non-small cell lung cancer (NSCLC) patients. In this study, we used high-throughput formalin-fixed and paraffin-embedded (FFPE) lung cancer tissue arrays and in situ hybridization (ISH) to determine the clinical significances of miRNA-148a and aimed to find novel target of miRNA-148a in lung cancer. Our results showed that there were 86 of 159 patients with low miRNA-148a expression and miRNA-148a was significantly down-regulated in primary cancer tissues when compared with their adjacent normal lung tissues. Low expression of miRNA-148a was strongly associated with high tumor grade, lymph node (LN) metastasis and a higher risk of tumor-related death in NSCLC. Lentivirus mediated overexpression of miRNA-148a inhibited migration and invasion of A549 and H1299 lung cancer cells. Furthermore, we validated Wnt1 as a direct target of miRNA-148a. Our data showed that the Wnt1 expression was negatively correlated with the expression of miRNA-148a in both primary cancer tissues and their corresponding adjacent normal lung tissues. In addition, overexpression of miRNA-148a inhibited Wnt1 protein expression in cancer cells. And knocking down of Wnt-1 by siRNA had the similar effect of miRNA-148a overexpression on cell migration and invasion in lung cancer cells. In conclusion, our results suggest that miRNA-148a inhibited cell migration and invasion through targeting Wnt1 and this might provide a new insight into the molecular mechanisms of lung cancer metastasis.</p></div