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Secreted miR-34a in astrocytic shedding vesicles enhanced the vulnerability of dopaminergic neurons to neurotoxins by targeting Bcl-2.

Mao S, Sun Q, Xiao H, Zhang C, Li L - Protein Cell (2015)

Bottom Line: To elucidate the potential role of glial MVs in disease, we evaluated the effects of secreted astrocytic MVs on stress condition.Further investigation showed that increased astrocytic miR-34a in SVs was involved in this progress via targeting anti-apoptotic protein Bcl-2 in dopaminergic neurons.These data revealed a novel mechanism underlying astrocyte-neuron interaction in disease.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Pharmaceutical Biotechnology, Nanjing Advanced Institute for Life Sciences (NAILS), Nanjing University School of Life Sciences, Nanjing, 210093, China.

ABSTRACT
MicroRNAs (miRNAs) are a class of noncoding RNAs that regulates target gene expression at posttranscriptional level, leading to further biological functions. We have demonstrated that microvesicles (MVs) can deliver miRNAs into target cells as a novel way of intercellular communication. It is reported that in central nervous system, glial cells release MVs, which modulate neuronal function in normal condition. To elucidate the potential role of glial MVs in disease, we evaluated the effects of secreted astrocytic MVs on stress condition. Our results demonstrated that after Lipopolysaccharide (LPS) stimulation, astrocytes released shedding vesicles (SVs) that enhanced vulnerability of dopaminergic neurons to neurotoxin. Further investigation showed that increased astrocytic miR-34a in SVs was involved in this progress via targeting anti-apoptotic protein Bcl-2 in dopaminergic neurons. We also found that inhibition of astrocytic miR-34a after LPS stimulation can postpone dopaminergic neuron loss under neurotoxin stress. These data revealed a novel mechanism underlying astrocyte-neuron interaction in disease.

No MeSH data available.


Related in: MedlinePlus

SVs derived from LPS-stimulated primary astrocytes enhance DA neurons loss after treatment with neurotoxins. (A and C) TH staining and quantification of the percentage of TH-positive cells among primary neurons pre-treated with different groups of SVs after 0.1 μmol/L MPP+ stress, *P < 0.05, **P < 0.01. Inset: DAPI staining of the cell nuclei in the field of view. (B and D) TH staining and quantification of the percentage of TH-positive cells among primary neurons pre-treated with different groups of SVs after 2 μmol/L 6-OHDA stress, *P < 0.05, **P < 0.01, scale bar = 200 μm. Inset: DAPI staining of the cell nuclei in the field of view. Control: a control group of neuron culture without SVs incubation; Control SV: SVs derived from primary astrocytes; LPS SV: SVs derived from LPS-stimulated primary astrocytes; anti-miR-34a + LPS SV: SVs derived from LPS-stimulated primary astrocytes transfected with miR-34a inhibitor; Sc antimiR + LPS SV: SVs derived from LPS-stimulated primary astrocytes transfected with scramble RNA; Sc antagomiR + LPS SV: SVs derived from LPS-stimulated primary astrocytes, and the primary neurons were pretreated with scramble antagomiR; antagomiR-34a + LPS SV: SVs derived from LPS-stimulated primary astrocytes, and the primary neurons were pretreated with antagomiR-34a
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Fig5: SVs derived from LPS-stimulated primary astrocytes enhance DA neurons loss after treatment with neurotoxins. (A and C) TH staining and quantification of the percentage of TH-positive cells among primary neurons pre-treated with different groups of SVs after 0.1 μmol/L MPP+ stress, *P < 0.05, **P < 0.01. Inset: DAPI staining of the cell nuclei in the field of view. (B and D) TH staining and quantification of the percentage of TH-positive cells among primary neurons pre-treated with different groups of SVs after 2 μmol/L 6-OHDA stress, *P < 0.05, **P < 0.01, scale bar = 200 μm. Inset: DAPI staining of the cell nuclei in the field of view. Control: a control group of neuron culture without SVs incubation; Control SV: SVs derived from primary astrocytes; LPS SV: SVs derived from LPS-stimulated primary astrocytes; anti-miR-34a + LPS SV: SVs derived from LPS-stimulated primary astrocytes transfected with miR-34a inhibitor; Sc antimiR + LPS SV: SVs derived from LPS-stimulated primary astrocytes transfected with scramble RNA; Sc antagomiR + LPS SV: SVs derived from LPS-stimulated primary astrocytes, and the primary neurons were pretreated with scramble antagomiR; antagomiR-34a + LPS SV: SVs derived from LPS-stimulated primary astrocytes, and the primary neurons were pretreated with antagomiR-34a

Mentions: Here, we further used primary culture to verify the results demonstrated in the cell lines. We collected LPS SVs from primary astrocytes and found that the miR-34a levels were also increased (Fig. S4). Pretreatment with primary astrocytic LPS SVs caused significant TH-positive cell loss after low concentration MPP+ (0.1 μmol/L) or 6-OHDA (2 μmol/L) treatment, without significant effect on the total neuron number showed by DAPI (Fig. 5A–D). This effect was strongly attenuated after blocking the up-regulation of miR-34a in astrocytic LPS SVs by anti-miR-34a pretreatment in astrocytes. To further verify that miR-34a is necessary and sufficient to induce apoptosis, we also used antagomiR oligonucleotides directed against miR-34a (antagomiR-34a) to block the level of miR-34a in primary neurons. Similar results were obtained (Fig. 5A–D). These results indicated that in an ex vivo situation, stressed astrocytes released SVs that increased TH neuron vulnerability, in which an increased level of secreted miR-34a may be involved.Figure 5


Secreted miR-34a in astrocytic shedding vesicles enhanced the vulnerability of dopaminergic neurons to neurotoxins by targeting Bcl-2.

Mao S, Sun Q, Xiao H, Zhang C, Li L - Protein Cell (2015)

SVs derived from LPS-stimulated primary astrocytes enhance DA neurons loss after treatment with neurotoxins. (A and C) TH staining and quantification of the percentage of TH-positive cells among primary neurons pre-treated with different groups of SVs after 0.1 μmol/L MPP+ stress, *P < 0.05, **P < 0.01. Inset: DAPI staining of the cell nuclei in the field of view. (B and D) TH staining and quantification of the percentage of TH-positive cells among primary neurons pre-treated with different groups of SVs after 2 μmol/L 6-OHDA stress, *P < 0.05, **P < 0.01, scale bar = 200 μm. Inset: DAPI staining of the cell nuclei in the field of view. Control: a control group of neuron culture without SVs incubation; Control SV: SVs derived from primary astrocytes; LPS SV: SVs derived from LPS-stimulated primary astrocytes; anti-miR-34a + LPS SV: SVs derived from LPS-stimulated primary astrocytes transfected with miR-34a inhibitor; Sc antimiR + LPS SV: SVs derived from LPS-stimulated primary astrocytes transfected with scramble RNA; Sc antagomiR + LPS SV: SVs derived from LPS-stimulated primary astrocytes, and the primary neurons were pretreated with scramble antagomiR; antagomiR-34a + LPS SV: SVs derived from LPS-stimulated primary astrocytes, and the primary neurons were pretreated with antagomiR-34a
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Fig5: SVs derived from LPS-stimulated primary astrocytes enhance DA neurons loss after treatment with neurotoxins. (A and C) TH staining and quantification of the percentage of TH-positive cells among primary neurons pre-treated with different groups of SVs after 0.1 μmol/L MPP+ stress, *P < 0.05, **P < 0.01. Inset: DAPI staining of the cell nuclei in the field of view. (B and D) TH staining and quantification of the percentage of TH-positive cells among primary neurons pre-treated with different groups of SVs after 2 μmol/L 6-OHDA stress, *P < 0.05, **P < 0.01, scale bar = 200 μm. Inset: DAPI staining of the cell nuclei in the field of view. Control: a control group of neuron culture without SVs incubation; Control SV: SVs derived from primary astrocytes; LPS SV: SVs derived from LPS-stimulated primary astrocytes; anti-miR-34a + LPS SV: SVs derived from LPS-stimulated primary astrocytes transfected with miR-34a inhibitor; Sc antimiR + LPS SV: SVs derived from LPS-stimulated primary astrocytes transfected with scramble RNA; Sc antagomiR + LPS SV: SVs derived from LPS-stimulated primary astrocytes, and the primary neurons were pretreated with scramble antagomiR; antagomiR-34a + LPS SV: SVs derived from LPS-stimulated primary astrocytes, and the primary neurons were pretreated with antagomiR-34a
Mentions: Here, we further used primary culture to verify the results demonstrated in the cell lines. We collected LPS SVs from primary astrocytes and found that the miR-34a levels were also increased (Fig. S4). Pretreatment with primary astrocytic LPS SVs caused significant TH-positive cell loss after low concentration MPP+ (0.1 μmol/L) or 6-OHDA (2 μmol/L) treatment, without significant effect on the total neuron number showed by DAPI (Fig. 5A–D). This effect was strongly attenuated after blocking the up-regulation of miR-34a in astrocytic LPS SVs by anti-miR-34a pretreatment in astrocytes. To further verify that miR-34a is necessary and sufficient to induce apoptosis, we also used antagomiR oligonucleotides directed against miR-34a (antagomiR-34a) to block the level of miR-34a in primary neurons. Similar results were obtained (Fig. 5A–D). These results indicated that in an ex vivo situation, stressed astrocytes released SVs that increased TH neuron vulnerability, in which an increased level of secreted miR-34a may be involved.Figure 5

Bottom Line: To elucidate the potential role of glial MVs in disease, we evaluated the effects of secreted astrocytic MVs on stress condition.Further investigation showed that increased astrocytic miR-34a in SVs was involved in this progress via targeting anti-apoptotic protein Bcl-2 in dopaminergic neurons.These data revealed a novel mechanism underlying astrocyte-neuron interaction in disease.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Pharmaceutical Biotechnology, Nanjing Advanced Institute for Life Sciences (NAILS), Nanjing University School of Life Sciences, Nanjing, 210093, China.

ABSTRACT
MicroRNAs (miRNAs) are a class of noncoding RNAs that regulates target gene expression at posttranscriptional level, leading to further biological functions. We have demonstrated that microvesicles (MVs) can deliver miRNAs into target cells as a novel way of intercellular communication. It is reported that in central nervous system, glial cells release MVs, which modulate neuronal function in normal condition. To elucidate the potential role of glial MVs in disease, we evaluated the effects of secreted astrocytic MVs on stress condition. Our results demonstrated that after Lipopolysaccharide (LPS) stimulation, astrocytes released shedding vesicles (SVs) that enhanced vulnerability of dopaminergic neurons to neurotoxin. Further investigation showed that increased astrocytic miR-34a in SVs was involved in this progress via targeting anti-apoptotic protein Bcl-2 in dopaminergic neurons. We also found that inhibition of astrocytic miR-34a after LPS stimulation can postpone dopaminergic neuron loss under neurotoxin stress. These data revealed a novel mechanism underlying astrocyte-neuron interaction in disease.

No MeSH data available.


Related in: MedlinePlus