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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

Co-incubation with LPS SVs increases miR-34a level while down-regulates Bcl-2 level that further compromises neurotoxin resistance of SH-SY5Y cells. (A) QPCR analysis of miR-34a levels in SH-SY5Y cells after co-incubation with different groups of SVs, **P < 0.01; (B and C) Western-blot and quantification of Bcl-2 protein levels in SH-SY5Y cells after co-incubation with different groups of SVs, *P < 0.05; (D and E) Viability of SH-SY5Y cells pre-treated with different groups of SVs following 0.2 mmol/L MPP+ or 10 μmol/L 6-OHDA stress, *P < 0.05; (F and G) TUNEL staining of SH-SY5Y cells pre-treated with different groups of SVs after 0.2 mmol/L MPP+ or 10 μmol/L 6-OHDA stress, scale bar = 50 μm; (H and I) Percentage of TUNEL positive cells among SH-SY5Y cells pre-treated with different types of SVs after 0.2 mmol/L MPP+ or 10 μmol/L 6-OHDA stress, *P < 0.05, **P < 0.01; Control SV: SVs derived from control U-87 MG cells; LPS SV: SVs derived from LPS-stimulated U-87 MG cells; anti miR-34a + LPS SV: SVs derived from LPS-stimulated U-87 MG cells transfected with miR-34a inhibitor; Scramble + LPS SV: SVs derived from LPS-stimulated U-87 MG cells transfected with scramble RNA
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Fig3: Co-incubation with LPS SVs increases miR-34a level while down-regulates Bcl-2 level that further compromises neurotoxin resistance of SH-SY5Y cells. (A) QPCR analysis of miR-34a levels in SH-SY5Y cells after co-incubation with different groups of SVs, **P < 0.01; (B and C) Western-blot and quantification of Bcl-2 protein levels in SH-SY5Y cells after co-incubation with different groups of SVs, *P < 0.05; (D and E) Viability of SH-SY5Y cells pre-treated with different groups of SVs following 0.2 mmol/L MPP+ or 10 μmol/L 6-OHDA stress, *P < 0.05; (F and G) TUNEL staining of SH-SY5Y cells pre-treated with different groups of SVs after 0.2 mmol/L MPP+ or 10 μmol/L 6-OHDA stress, scale bar = 50 μm; (H and I) Percentage of TUNEL positive cells among SH-SY5Y cells pre-treated with different types of SVs after 0.2 mmol/L MPP+ or 10 μmol/L 6-OHDA stress, *P < 0.05, **P < 0.01; Control SV: SVs derived from control U-87 MG cells; LPS SV: SVs derived from LPS-stimulated U-87 MG cells; anti miR-34a + LPS SV: SVs derived from LPS-stimulated U-87 MG cells transfected with miR-34a inhibitor; Scramble + LPS SV: SVs derived from LPS-stimulated U-87 MG cells transfected with scramble RNA

Mentions: As it has been reported that miR-34a may target Bcl-2 protein, we first performed the luciferase assay and Western blot in SH-SY5Y cells which showed that miR-34a can indeed target Bcl-2 mRNA directly and repress Bcl-2 protein levels in SH-SY5Y cells (Fig. S2). Next, we further investigated the role of secreted miR-34a in neuronal loss under neurotoxin treatment. We found that the level of mature miR-34a was significantly increased in SH-SY5Y cells after the treatment with LPS SVs (Fig. 3A). As a result, the protein level of Bcl-2 was repressed in these cells (Fig. 3B and 3C). However, pretreatment with anti-miR-34a in U-87 MG cells blocked the increase of miR-34a and negated the repression of Bcl-2 caused by LPS SVs (Fig. 3A–C). Further investigation showed that cellular viability was reduced and apoptotic ratio was increased in LPS SV-treated SH-SY5Y cells after 0.2 mmol/L MPP+ exposure. On the other hand, pretreating U-87 MG cells with anti-miR-34a significantly lessened the effect of LPS SVs on SH-SY5Y cell viability and apoptotic ratio (Fig. 3D, 3F and 3H). Similar results were achieved when 6-OHDA was used (Fig. 3E, 3G and 3I).Figure 3


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)

Co-incubation with LPS SVs increases miR-34a level while down-regulates Bcl-2 level that further compromises neurotoxin resistance of SH-SY5Y cells. (A) QPCR analysis of miR-34a levels in SH-SY5Y cells after co-incubation with different groups of SVs, **P < 0.01; (B and C) Western-blot and quantification of Bcl-2 protein levels in SH-SY5Y cells after co-incubation with different groups of SVs, *P < 0.05; (D and E) Viability of SH-SY5Y cells pre-treated with different groups of SVs following 0.2 mmol/L MPP+ or 10 μmol/L 6-OHDA stress, *P < 0.05; (F and G) TUNEL staining of SH-SY5Y cells pre-treated with different groups of SVs after 0.2 mmol/L MPP+ or 10 μmol/L 6-OHDA stress, scale bar = 50 μm; (H and I) Percentage of TUNEL positive cells among SH-SY5Y cells pre-treated with different types of SVs after 0.2 mmol/L MPP+ or 10 μmol/L 6-OHDA stress, *P < 0.05, **P < 0.01; Control SV: SVs derived from control U-87 MG cells; LPS SV: SVs derived from LPS-stimulated U-87 MG cells; anti miR-34a + LPS SV: SVs derived from LPS-stimulated U-87 MG cells transfected with miR-34a inhibitor; Scramble + LPS SV: SVs derived from LPS-stimulated U-87 MG cells transfected with scramble RNA
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Related In: Results  -  Collection

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Fig3: Co-incubation with LPS SVs increases miR-34a level while down-regulates Bcl-2 level that further compromises neurotoxin resistance of SH-SY5Y cells. (A) QPCR analysis of miR-34a levels in SH-SY5Y cells after co-incubation with different groups of SVs, **P < 0.01; (B and C) Western-blot and quantification of Bcl-2 protein levels in SH-SY5Y cells after co-incubation with different groups of SVs, *P < 0.05; (D and E) Viability of SH-SY5Y cells pre-treated with different groups of SVs following 0.2 mmol/L MPP+ or 10 μmol/L 6-OHDA stress, *P < 0.05; (F and G) TUNEL staining of SH-SY5Y cells pre-treated with different groups of SVs after 0.2 mmol/L MPP+ or 10 μmol/L 6-OHDA stress, scale bar = 50 μm; (H and I) Percentage of TUNEL positive cells among SH-SY5Y cells pre-treated with different types of SVs after 0.2 mmol/L MPP+ or 10 μmol/L 6-OHDA stress, *P < 0.05, **P < 0.01; Control SV: SVs derived from control U-87 MG cells; LPS SV: SVs derived from LPS-stimulated U-87 MG cells; anti miR-34a + LPS SV: SVs derived from LPS-stimulated U-87 MG cells transfected with miR-34a inhibitor; Scramble + LPS SV: SVs derived from LPS-stimulated U-87 MG cells transfected with scramble RNA
Mentions: As it has been reported that miR-34a may target Bcl-2 protein, we first performed the luciferase assay and Western blot in SH-SY5Y cells which showed that miR-34a can indeed target Bcl-2 mRNA directly and repress Bcl-2 protein levels in SH-SY5Y cells (Fig. S2). Next, we further investigated the role of secreted miR-34a in neuronal loss under neurotoxin treatment. We found that the level of mature miR-34a was significantly increased in SH-SY5Y cells after the treatment with LPS SVs (Fig. 3A). As a result, the protein level of Bcl-2 was repressed in these cells (Fig. 3B and 3C). However, pretreatment with anti-miR-34a in U-87 MG cells blocked the increase of miR-34a and negated the repression of Bcl-2 caused by LPS SVs (Fig. 3A–C). Further investigation showed that cellular viability was reduced and apoptotic ratio was increased in LPS SV-treated SH-SY5Y cells after 0.2 mmol/L MPP+ exposure. On the other hand, pretreating U-87 MG cells with anti-miR-34a significantly lessened the effect of LPS SVs on SH-SY5Y cell viability and apoptotic ratio (Fig. 3D, 3F and 3H). Similar results were achieved when 6-OHDA was used (Fig. 3E, 3G and 3I).Figure 3

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