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Up-regulation of neural and cell cycle-related microRNAs in brain of amyotrophic lateral sclerosis mice at late disease stage.

Marcuzzo S, Bonanno S, Kapetis D, Barzago C, Cavalcante P, D'Alessandro S, Mantegazza R, Bernasconi P - Mol Brain (2015)

Bottom Line: Expression of miR-9, miR-124a, miR-19a and -19b was significantly increased in G93A-SOD1 whole brain at late stage disease compared to B6.SJL and Wt-SOD1 control brains.In G93A-SOD1 brainstem motor nuclei and primary motor cortex, miR-9 and miR-124a were significantly up-regulated, miR-125b expression was also increased. miR-19a and -19b were up-regulated in primary motor cortex and hippocampus, respectively.Expression analysis of predicted miRNA targets identified miRNA/target gene pairs differentially expressed in G93A-SOD1 brain regions compared to controls.

View Article: PubMed Central - PubMed

Affiliation: Neurology IV - Neuromuscular Diseases and Neuroimmunology Unit, Fondazione Istituto Neurologico "Carlo Besta", Via Celoria 11, Milan, 20133, Italy. stefania.marcuzzo@istituto-besta.it.

ABSTRACT

Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motor neuron degeneration in motor cortex, brainstem and spinal cord. microRNAs (miRNAs) are small non-coding RNAs that bind complementary target sequences and modulate gene expression; they are key molecules for establishing a neuronal phenotype, and in neurodegeneration. Here we investigated neural miR-9, miR-124a, miR-125b, miR-219, miR-134, and cell cycle-related miR-19a and -19b, in G93A-SOD1 mouse brain in pre-symptomatic and late stage disease.

Results: Expression of miR-9, miR-124a, miR-19a and -19b was significantly increased in G93A-SOD1 whole brain at late stage disease compared to B6.SJL and Wt-SOD1 control brains. These miRNAs were then analyzed in manually dissected SVZ, hippocampus, primary motor cortex and brainstem motor nuclei in 18-week-old ALS mice compared to same age controls. In SVZ and hippocampus miR-124a was up-regulated, miR-219 was down-regulated, and numbers of neural stem progenitor cells (NSPCs) were significantly increased. In G93A-SOD1 brainstem motor nuclei and primary motor cortex, miR-9 and miR-124a were significantly up-regulated, miR-125b expression was also increased. miR-19a and -19b were up-regulated in primary motor cortex and hippocampus, respectively. Expression analysis of predicted miRNA targets identified miRNA/target gene pairs differentially expressed in G93A-SOD1 brain regions compared to controls.

Conclusions: Hierarchical clustering analysis, identifying two clusters of miRNA/target genes, one characterizing brainstem motor nuclei and primary motor cortex, the other hippocampus and SVZ, suggests that altered expression of neural and cell cycle-related miRNAs in these brain regions might contribute to ALS pathogenesis in G93A-SOD1 mice. Re-establishing their expression to normal levels could be a new therapeutic approach to ALS.

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Increased numbers of differentiating NSPCs in SVZ of G93A-SOD1 brain. (A) Confocal microscopy images of dorsal and ventral regions of SVZ in G93A-SOD1 and Wt-SOD1 brain at postnatal week 18, stained for nestin (green), GFAP (red), and vimentin (blue). Scale bar = 50 μm. (B) Quantification of nestin-, GFAP-, and vimentin-positive cells in SVZ of G93A-SOD1 and Wt-SOD1 mice. Data are means ± SD of 3 mice per group. **p < 0.001; limma moderated t-test. (C) Hematoxylin and eosin staining of SVZ sections adjacent to those analyzed by confocal microscopy. Scale bar = 50 μm. D: dorsal. V: ventral. (D) Confocal microscopy images showing Dlx2-stained cells in ventral SVZ in G93A-SOD1 and Wt-SOD1 brain. Scale bar = 50 μm. (E) Quantification of Dlx2-positive cells in G93A-SOD1 and Wt-SOD1 SVZ. Data are means ± SD of 3 mice per group. **p < 0.001; limma moderated t-test.
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Fig4: Increased numbers of differentiating NSPCs in SVZ of G93A-SOD1 brain. (A) Confocal microscopy images of dorsal and ventral regions of SVZ in G93A-SOD1 and Wt-SOD1 brain at postnatal week 18, stained for nestin (green), GFAP (red), and vimentin (blue). Scale bar = 50 μm. (B) Quantification of nestin-, GFAP-, and vimentin-positive cells in SVZ of G93A-SOD1 and Wt-SOD1 mice. Data are means ± SD of 3 mice per group. **p < 0.001; limma moderated t-test. (C) Hematoxylin and eosin staining of SVZ sections adjacent to those analyzed by confocal microscopy. Scale bar = 50 μm. D: dorsal. V: ventral. (D) Confocal microscopy images showing Dlx2-stained cells in ventral SVZ in G93A-SOD1 and Wt-SOD1 brain. Scale bar = 50 μm. (E) Quantification of Dlx2-positive cells in G93A-SOD1 and Wt-SOD1 SVZ. Data are means ± SD of 3 mice per group. **p < 0.001; limma moderated t-test.

Mentions: The SVZ is the primary source of new neurons in adult brain. Neurons arise from unipolar bipolar type B stem cells that express nestin and vimentin and/or glial fibrillary acidic protein (GFAP): they give rise to transit-amplifying type C cells, and in turn produce type A neuroblasts expressing Dlx2 – which are migratory and proliferative neuronal precursors [8]. Immunohistochemical analysis for nestin, GFAP and vimentin revealed significantly more unipolar and bipolar cells positive for these markers in the dorsal and ventral SVZ of 18-week-old ALS mice compared with same areas in Wt-SOD1 mice (p < 0.001; Figure 4A-C). We also found significantly more Dlx2-positive cells in the ventral SVZ of ALS than Wt-SOD1 mice (p < 0.001; Figure 4D, E). These findings suggest that neurogenesis is occurring in the SVZ of ALS mice at late stage disease. In the adult mammalian brain neurogenesis also occurs in the hippocampal dentate gyrus [38]. Immunohistochemical analysis of hippocampal dentate gyrus revealed significantly more nestin- and GFAP-positive NSPCs in 18-week-old ALS mice than same-age controls (p < 0.05; Figure 5).Figure 4


Up-regulation of neural and cell cycle-related microRNAs in brain of amyotrophic lateral sclerosis mice at late disease stage.

Marcuzzo S, Bonanno S, Kapetis D, Barzago C, Cavalcante P, D'Alessandro S, Mantegazza R, Bernasconi P - Mol Brain (2015)

Increased numbers of differentiating NSPCs in SVZ of G93A-SOD1 brain. (A) Confocal microscopy images of dorsal and ventral regions of SVZ in G93A-SOD1 and Wt-SOD1 brain at postnatal week 18, stained for nestin (green), GFAP (red), and vimentin (blue). Scale bar = 50 μm. (B) Quantification of nestin-, GFAP-, and vimentin-positive cells in SVZ of G93A-SOD1 and Wt-SOD1 mice. Data are means ± SD of 3 mice per group. **p < 0.001; limma moderated t-test. (C) Hematoxylin and eosin staining of SVZ sections adjacent to those analyzed by confocal microscopy. Scale bar = 50 μm. D: dorsal. V: ventral. (D) Confocal microscopy images showing Dlx2-stained cells in ventral SVZ in G93A-SOD1 and Wt-SOD1 brain. Scale bar = 50 μm. (E) Quantification of Dlx2-positive cells in G93A-SOD1 and Wt-SOD1 SVZ. Data are means ± SD of 3 mice per group. **p < 0.001; limma moderated t-test.
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Fig4: Increased numbers of differentiating NSPCs in SVZ of G93A-SOD1 brain. (A) Confocal microscopy images of dorsal and ventral regions of SVZ in G93A-SOD1 and Wt-SOD1 brain at postnatal week 18, stained for nestin (green), GFAP (red), and vimentin (blue). Scale bar = 50 μm. (B) Quantification of nestin-, GFAP-, and vimentin-positive cells in SVZ of G93A-SOD1 and Wt-SOD1 mice. Data are means ± SD of 3 mice per group. **p < 0.001; limma moderated t-test. (C) Hematoxylin and eosin staining of SVZ sections adjacent to those analyzed by confocal microscopy. Scale bar = 50 μm. D: dorsal. V: ventral. (D) Confocal microscopy images showing Dlx2-stained cells in ventral SVZ in G93A-SOD1 and Wt-SOD1 brain. Scale bar = 50 μm. (E) Quantification of Dlx2-positive cells in G93A-SOD1 and Wt-SOD1 SVZ. Data are means ± SD of 3 mice per group. **p < 0.001; limma moderated t-test.
Mentions: The SVZ is the primary source of new neurons in adult brain. Neurons arise from unipolar bipolar type B stem cells that express nestin and vimentin and/or glial fibrillary acidic protein (GFAP): they give rise to transit-amplifying type C cells, and in turn produce type A neuroblasts expressing Dlx2 – which are migratory and proliferative neuronal precursors [8]. Immunohistochemical analysis for nestin, GFAP and vimentin revealed significantly more unipolar and bipolar cells positive for these markers in the dorsal and ventral SVZ of 18-week-old ALS mice compared with same areas in Wt-SOD1 mice (p < 0.001; Figure 4A-C). We also found significantly more Dlx2-positive cells in the ventral SVZ of ALS than Wt-SOD1 mice (p < 0.001; Figure 4D, E). These findings suggest that neurogenesis is occurring in the SVZ of ALS mice at late stage disease. In the adult mammalian brain neurogenesis also occurs in the hippocampal dentate gyrus [38]. Immunohistochemical analysis of hippocampal dentate gyrus revealed significantly more nestin- and GFAP-positive NSPCs in 18-week-old ALS mice than same-age controls (p < 0.05; Figure 5).Figure 4

Bottom Line: Expression of miR-9, miR-124a, miR-19a and -19b was significantly increased in G93A-SOD1 whole brain at late stage disease compared to B6.SJL and Wt-SOD1 control brains.In G93A-SOD1 brainstem motor nuclei and primary motor cortex, miR-9 and miR-124a were significantly up-regulated, miR-125b expression was also increased. miR-19a and -19b were up-regulated in primary motor cortex and hippocampus, respectively.Expression analysis of predicted miRNA targets identified miRNA/target gene pairs differentially expressed in G93A-SOD1 brain regions compared to controls.

View Article: PubMed Central - PubMed

Affiliation: Neurology IV - Neuromuscular Diseases and Neuroimmunology Unit, Fondazione Istituto Neurologico "Carlo Besta", Via Celoria 11, Milan, 20133, Italy. stefania.marcuzzo@istituto-besta.it.

ABSTRACT

Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motor neuron degeneration in motor cortex, brainstem and spinal cord. microRNAs (miRNAs) are small non-coding RNAs that bind complementary target sequences and modulate gene expression; they are key molecules for establishing a neuronal phenotype, and in neurodegeneration. Here we investigated neural miR-9, miR-124a, miR-125b, miR-219, miR-134, and cell cycle-related miR-19a and -19b, in G93A-SOD1 mouse brain in pre-symptomatic and late stage disease.

Results: Expression of miR-9, miR-124a, miR-19a and -19b was significantly increased in G93A-SOD1 whole brain at late stage disease compared to B6.SJL and Wt-SOD1 control brains. These miRNAs were then analyzed in manually dissected SVZ, hippocampus, primary motor cortex and brainstem motor nuclei in 18-week-old ALS mice compared to same age controls. In SVZ and hippocampus miR-124a was up-regulated, miR-219 was down-regulated, and numbers of neural stem progenitor cells (NSPCs) were significantly increased. In G93A-SOD1 brainstem motor nuclei and primary motor cortex, miR-9 and miR-124a were significantly up-regulated, miR-125b expression was also increased. miR-19a and -19b were up-regulated in primary motor cortex and hippocampus, respectively. Expression analysis of predicted miRNA targets identified miRNA/target gene pairs differentially expressed in G93A-SOD1 brain regions compared to controls.

Conclusions: Hierarchical clustering analysis, identifying two clusters of miRNA/target genes, one characterizing brainstem motor nuclei and primary motor cortex, the other hippocampus and SVZ, suggests that altered expression of neural and cell cycle-related miRNAs in these brain regions might contribute to ALS pathogenesis in G93A-SOD1 mice. Re-establishing their expression to normal levels could be a new therapeutic approach to ALS.

Show MeSH
Related in: MedlinePlus