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Role of astroglia in Down's syndrome revealed by patient-derived human-induced pluripotent stem cells.

Chen C, Jiang P, Xue H, Peterson SE, Tran HT, McCann AE, Parast MM, Li S, Pleasure DE, Laurent LC, Loring JF, Liu Y, Deng W - Nat Commun (2014)

Bottom Line: DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules.Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo.Finally, we show that the FDA-approved antibiotic drug, minocycline, partially corrects the pathological phenotypes of DS astroglia by specifically modulating the expression of S100B, GFAP, inducible nitric oxide synthase, and thrombospondins 1 and 2 in DS astroglia.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, California 95817, USA [2] Institute for Pediatric Regenerative Medicine, Shriners Hospitals for Children, Sacramento, California 95817, USA [3] Department of Neurology, Institute of Neurology, Tianjin General Hospital, Tianjin Medical University, Tianjin 300070, China [4].

ABSTRACT
Down's syndrome (DS), caused by trisomy of human chromosome 21, is the most common genetic cause of intellectual disability. Here we use induced pluripotent stem cells (iPSCs) derived from DS patients to identify a role for astrocytes in DS pathogenesis. DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules. Astrocyte-conditioned medium collected from DS astroglia causes toxicity to neurons, and fails to promote neuronal ion channel maturation and synapse formation. Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo. We also observed abnormal gene expression profiles from DS astroglia. Finally, we show that the FDA-approved antibiotic drug, minocycline, partially corrects the pathological phenotypes of DS astroglia by specifically modulating the expression of S100B, GFAP, inducible nitric oxide synthase, and thrombospondins 1 and 2 in DS astroglia. Our studies shed light on the pathogenesis and possible treatment of DS by targeting astrocytes with a clinically available drug.

No MeSH data available.


Related in: MedlinePlus

Identification and correction of pathological phenotypes of DSastroglia.(a1–6) qPCR analysis of S100B, GFAP, iNOS, TSP-1, TSP-2 and NFE2L2 mRNA expression inDS and control (Cont) astroglia. (a7) Quantification of nitrite/nitrate concentration in ACMcollected from DS and Cont astroglia. One-way analysis of variance (ANOVA)test, ♣P<0.05, ♣♣P<0.01 and♣♣♣P<0.001, comparison between two DS astroglia with Cont 1astroglia. #P<0.05,##P<0.01 and###P<0.001, comparison between two DS astrogliawith Cont 2 astroglia. Student’s t-test, *P<0.05, *P<0.01 and *♣P<0.001, comparison between two DS astroglia or twoCont astroglia. n=3–4 for each cell line. (b)Representative and quantification of ROS production in Cont and DSastroglia. Green fluorescence marks cells that undergo oxidation.Student’s t-test, **P<0.01.n=3–4 from each cell line. (c) Quantification ofpooled data showing the proliferation rate of Cont and DS astroglia.Student’s t-test, **P<0.01.n=3–4 from each cell line. (d) Glutamate uptake analysis showingthat both DS and Cont astroglia were capable of glutamate uptake. Notice that DSastroglia show glutamateuptake at a higher rate at the 30- and 60-min time point than Contastroglia. Student’s t-test, **P<0.01 and***P<0.001, n=4 from each cell line. (e)Representatives showing intracellular uptake of the BLOCK-iT FluorescentOligo at 24 h after transfection of DS astroglia. Scale bar,50 μm. (f) qPCR analysis of pooled data showingthe expression of S100B gene in DS astroglia at 48 hafter transfection with Cont and S100B siRNA. Student’s t-test,**P<0.01, n=3–5 from each cell line.(g) Representatives and quantification of pooled data showing ROSproduction in DS astroglia determined at 48 h after transfectionwith Cont and S100B siRNA. Student’s t-test,*P<0.05, n=5 from each cell line.(h,i) Quantification of pooled data showing theconcentration of nitrite/nitrate in the ACM and proliferation rate of DSastroglia at 48 h after transfection with Cont and S100B siRNA. Student’st-test, *P<0.05, n=3–4 from eachcell line. (j) qPCR analysis of S100B, iNOS and  NEF2L2mRNA expression in DS1 astroglia after the treatment of resveratrol, cucurmin or minocycline for 72 h.One-way ANOVA test, *P<0.05, **P<0.01 and***P<0.001; n=3–5 for each group.(k) Quantitative analysis of the proliferation rate of DS1 and 2astroglia after the treatment of minocycline. Student’s t-test,*P<0.05, n=3–4 for each cell line.(l) qPCR analysis of GFAP, TSP-1 and TSP-2 mRNA expression in DS astroglia after thetreatment of minocycline.Student’s t-test, *P<0.05 and**P<0.01; n=3–4 for each group. Data arepresented as mean±s.e.m.
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f2: Identification and correction of pathological phenotypes of DSastroglia.(a1–6) qPCR analysis of S100B, GFAP, iNOS, TSP-1, TSP-2 and NFE2L2 mRNA expression inDS and control (Cont) astroglia. (a7) Quantification of nitrite/nitrate concentration in ACMcollected from DS and Cont astroglia. One-way analysis of variance (ANOVA)test, ♣P<0.05, ♣♣P<0.01 and♣♣♣P<0.001, comparison between two DS astroglia with Cont 1astroglia. #P<0.05,##P<0.01 and###P<0.001, comparison between two DS astrogliawith Cont 2 astroglia. Student’s t-test, *P<0.05, *P<0.01 and *♣P<0.001, comparison between two DS astroglia or twoCont astroglia. n=3–4 for each cell line. (b)Representative and quantification of ROS production in Cont and DSastroglia. Green fluorescence marks cells that undergo oxidation.Student’s t-test, **P<0.01.n=3–4 from each cell line. (c) Quantification ofpooled data showing the proliferation rate of Cont and DS astroglia.Student’s t-test, **P<0.01.n=3–4 from each cell line. (d) Glutamate uptake analysis showingthat both DS and Cont astroglia were capable of glutamate uptake. Notice that DSastroglia show glutamateuptake at a higher rate at the 30- and 60-min time point than Contastroglia. Student’s t-test, **P<0.01 and***P<0.001, n=4 from each cell line. (e)Representatives showing intracellular uptake of the BLOCK-iT FluorescentOligo at 24 h after transfection of DS astroglia. Scale bar,50 μm. (f) qPCR analysis of pooled data showingthe expression of S100B gene in DS astroglia at 48 hafter transfection with Cont and S100B siRNA. Student’s t-test,**P<0.01, n=3–5 from each cell line.(g) Representatives and quantification of pooled data showing ROSproduction in DS astroglia determined at 48 h after transfectionwith Cont and S100B siRNA. Student’s t-test,*P<0.05, n=5 from each cell line.(h,i) Quantification of pooled data showing theconcentration of nitrite/nitrate in the ACM and proliferation rate of DSastroglia at 48 h after transfection with Cont and S100B siRNA. Student’st-test, *P<0.05, n=3–4 from eachcell line. (j) qPCR analysis of S100B, iNOS and NEF2L2mRNA expression in DS1 astroglia after the treatment of resveratrol, cucurmin or minocycline for 72 h.One-way ANOVA test, *P<0.05, **P<0.01 and***P<0.001; n=3–5 for each group.(k) Quantitative analysis of the proliferation rate of DS1 and 2astroglia after the treatment of minocycline. Student’s t-test,*P<0.05, n=3–4 for each cell line.(l) qPCR analysis of GFAP, TSP-1 and TSP-2 mRNA expression in DS astroglia after thetreatment of minocycline.Student’s t-test, *P<0.05 and**P<0.01; n=3–4 for each group. Data arepresented as mean±s.e.m.

Mentions: We further hypothesized that DS astroglia might critically contribute to theaforementioned abnormal DS NPC differentiation and the abnormal maturation of DSneurons. To test this hypothesis, we first examined the gene expression in theDS and control astroglia by qPCR. S100B is an astrocyte marker, and the human S100B gene maps to HSA21 and istriplicated in DS. Consistently, we found that DS astroglia expressed muchhigher level of S100Bthan control astroglia (Fig. 2a1). GFAP was also expressed at ahigher level in DS astroglia (Fig. 2a2), which isconsistent with previous observations of elevated expression of GFAP in the brain of Ts65Dn mouse, amouse model for DS25. To validate these findings, we alsoexamined the expression of S100B and GFAP in postmortem human brain tissue by immunostaining. Theimmunoreactivity of GFAP andS100B was much stronger inthe brain tissues of DS patients than in the normal controls. Moreover,astrocytes in the DS brain tissues exhibited an activated morphology, with morebranching and thicker branches, compared with astrocytes in the normal braintissues (Supplementary Fig. 3).Previous reports showed that overexpression of S100B induced the expression ofnitric oxide synthase(iNOS) and stimulatednitric oxide(NO) generation fromastrocytes26. Consistently, we observed that iNOS was expressed at a higherlevel in DS astroglia than in control astroglia (Fig.2a3). To confirm our findings, we maintained the astroglia in minimalmedium, and factors they secreted were collected as astroglial cell-conditionedmedium (ACM). As shown in Fig. 2a7, the ACM collected fromDS astroglia (DS ACM) contained higher concentration of nitrite/nitrate than the ACM collected fromcontrol astroglia (control ACM). Moreover, we examined the expression ofNFE2L2, thegene encodes erythroid 2-related factor2 (Nrf2),activation of which induces production of glutathione and confers astroglia non-cell-autonomousneuroprotective effects on neurons against oxidative insult2728, and the expression of genes encoding the secreted factors thrombospondins 1and 2 (TSP-1 and TSP-2), which are known to promotesynapse formation of neurons29. Interestingly, DS astrogliaexpressed lower levels of NFE2L2, TSP-1 and TSP-2 (Fig. 2a4), indicatingtheir potentially compromised neuroprotective and neurotrophic properties. Notethat variability of neural differentiation of different hiPSC lines wasreported30. We showed that variations were found within theDS astroglia and control astroglia derived from different hiPSCs (Fig. 2a). For example, compared with the control astroglia, DS1astroglia had a prominent increase of S100B gene expression (>74-fold), whereas DS2had a smaller increase (~2.5-fold). To ensure the reliability of ourobservations, each sample was prepared in triplicate and experiments wererepeated for at least three times. Conclusions were drawn only when each DS lineshowed the same trends of change with statistical significance when comparedwith each control line.


Role of astroglia in Down's syndrome revealed by patient-derived human-induced pluripotent stem cells.

Chen C, Jiang P, Xue H, Peterson SE, Tran HT, McCann AE, Parast MM, Li S, Pleasure DE, Laurent LC, Loring JF, Liu Y, Deng W - Nat Commun (2014)

Identification and correction of pathological phenotypes of DSastroglia.(a1–6) qPCR analysis of S100B, GFAP, iNOS, TSP-1, TSP-2 and NFE2L2 mRNA expression inDS and control (Cont) astroglia. (a7) Quantification of nitrite/nitrate concentration in ACMcollected from DS and Cont astroglia. One-way analysis of variance (ANOVA)test, ♣P<0.05, ♣♣P<0.01 and♣♣♣P<0.001, comparison between two DS astroglia with Cont 1astroglia. #P<0.05,##P<0.01 and###P<0.001, comparison between two DS astrogliawith Cont 2 astroglia. Student’s t-test, *P<0.05, *P<0.01 and *♣P<0.001, comparison between two DS astroglia or twoCont astroglia. n=3–4 for each cell line. (b)Representative and quantification of ROS production in Cont and DSastroglia. Green fluorescence marks cells that undergo oxidation.Student’s t-test, **P<0.01.n=3–4 from each cell line. (c) Quantification ofpooled data showing the proliferation rate of Cont and DS astroglia.Student’s t-test, **P<0.01.n=3–4 from each cell line. (d) Glutamate uptake analysis showingthat both DS and Cont astroglia were capable of glutamate uptake. Notice that DSastroglia show glutamateuptake at a higher rate at the 30- and 60-min time point than Contastroglia. Student’s t-test, **P<0.01 and***P<0.001, n=4 from each cell line. (e)Representatives showing intracellular uptake of the BLOCK-iT FluorescentOligo at 24 h after transfection of DS astroglia. Scale bar,50 μm. (f) qPCR analysis of pooled data showingthe expression of S100B gene in DS astroglia at 48 hafter transfection with Cont and S100B siRNA. Student’s t-test,**P<0.01, n=3–5 from each cell line.(g) Representatives and quantification of pooled data showing ROSproduction in DS astroglia determined at 48 h after transfectionwith Cont and S100B siRNA. Student’s t-test,*P<0.05, n=5 from each cell line.(h,i) Quantification of pooled data showing theconcentration of nitrite/nitrate in the ACM and proliferation rate of DSastroglia at 48 h after transfection with Cont and S100B siRNA. Student’st-test, *P<0.05, n=3–4 from eachcell line. (j) qPCR analysis of S100B, iNOS and  NEF2L2mRNA expression in DS1 astroglia after the treatment of resveratrol, cucurmin or minocycline for 72 h.One-way ANOVA test, *P<0.05, **P<0.01 and***P<0.001; n=3–5 for each group.(k) Quantitative analysis of the proliferation rate of DS1 and 2astroglia after the treatment of minocycline. Student’s t-test,*P<0.05, n=3–4 for each cell line.(l) qPCR analysis of GFAP, TSP-1 and TSP-2 mRNA expression in DS astroglia after thetreatment of minocycline.Student’s t-test, *P<0.05 and**P<0.01; n=3–4 for each group. Data arepresented as mean±s.e.m.
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f2: Identification and correction of pathological phenotypes of DSastroglia.(a1–6) qPCR analysis of S100B, GFAP, iNOS, TSP-1, TSP-2 and NFE2L2 mRNA expression inDS and control (Cont) astroglia. (a7) Quantification of nitrite/nitrate concentration in ACMcollected from DS and Cont astroglia. One-way analysis of variance (ANOVA)test, ♣P<0.05, ♣♣P<0.01 and♣♣♣P<0.001, comparison between two DS astroglia with Cont 1astroglia. #P<0.05,##P<0.01 and###P<0.001, comparison between two DS astrogliawith Cont 2 astroglia. Student’s t-test, *P<0.05, *P<0.01 and *♣P<0.001, comparison between two DS astroglia or twoCont astroglia. n=3–4 for each cell line. (b)Representative and quantification of ROS production in Cont and DSastroglia. Green fluorescence marks cells that undergo oxidation.Student’s t-test, **P<0.01.n=3–4 from each cell line. (c) Quantification ofpooled data showing the proliferation rate of Cont and DS astroglia.Student’s t-test, **P<0.01.n=3–4 from each cell line. (d) Glutamate uptake analysis showingthat both DS and Cont astroglia were capable of glutamate uptake. Notice that DSastroglia show glutamateuptake at a higher rate at the 30- and 60-min time point than Contastroglia. Student’s t-test, **P<0.01 and***P<0.001, n=4 from each cell line. (e)Representatives showing intracellular uptake of the BLOCK-iT FluorescentOligo at 24 h after transfection of DS astroglia. Scale bar,50 μm. (f) qPCR analysis of pooled data showingthe expression of S100B gene in DS astroglia at 48 hafter transfection with Cont and S100B siRNA. Student’s t-test,**P<0.01, n=3–5 from each cell line.(g) Representatives and quantification of pooled data showing ROSproduction in DS astroglia determined at 48 h after transfectionwith Cont and S100B siRNA. Student’s t-test,*P<0.05, n=5 from each cell line.(h,i) Quantification of pooled data showing theconcentration of nitrite/nitrate in the ACM and proliferation rate of DSastroglia at 48 h after transfection with Cont and S100B siRNA. Student’st-test, *P<0.05, n=3–4 from eachcell line. (j) qPCR analysis of S100B, iNOS and NEF2L2mRNA expression in DS1 astroglia after the treatment of resveratrol, cucurmin or minocycline for 72 h.One-way ANOVA test, *P<0.05, **P<0.01 and***P<0.001; n=3–5 for each group.(k) Quantitative analysis of the proliferation rate of DS1 and 2astroglia after the treatment of minocycline. Student’s t-test,*P<0.05, n=3–4 for each cell line.(l) qPCR analysis of GFAP, TSP-1 and TSP-2 mRNA expression in DS astroglia after thetreatment of minocycline.Student’s t-test, *P<0.05 and**P<0.01; n=3–4 for each group. Data arepresented as mean±s.e.m.
Mentions: We further hypothesized that DS astroglia might critically contribute to theaforementioned abnormal DS NPC differentiation and the abnormal maturation of DSneurons. To test this hypothesis, we first examined the gene expression in theDS and control astroglia by qPCR. S100B is an astrocyte marker, and the human S100B gene maps to HSA21 and istriplicated in DS. Consistently, we found that DS astroglia expressed muchhigher level of S100Bthan control astroglia (Fig. 2a1). GFAP was also expressed at ahigher level in DS astroglia (Fig. 2a2), which isconsistent with previous observations of elevated expression of GFAP in the brain of Ts65Dn mouse, amouse model for DS25. To validate these findings, we alsoexamined the expression of S100B and GFAP in postmortem human brain tissue by immunostaining. Theimmunoreactivity of GFAP andS100B was much stronger inthe brain tissues of DS patients than in the normal controls. Moreover,astrocytes in the DS brain tissues exhibited an activated morphology, with morebranching and thicker branches, compared with astrocytes in the normal braintissues (Supplementary Fig. 3).Previous reports showed that overexpression of S100B induced the expression ofnitric oxide synthase(iNOS) and stimulatednitric oxide(NO) generation fromastrocytes26. Consistently, we observed that iNOS was expressed at a higherlevel in DS astroglia than in control astroglia (Fig.2a3). To confirm our findings, we maintained the astroglia in minimalmedium, and factors they secreted were collected as astroglial cell-conditionedmedium (ACM). As shown in Fig. 2a7, the ACM collected fromDS astroglia (DS ACM) contained higher concentration of nitrite/nitrate than the ACM collected fromcontrol astroglia (control ACM). Moreover, we examined the expression ofNFE2L2, thegene encodes erythroid 2-related factor2 (Nrf2),activation of which induces production of glutathione and confers astroglia non-cell-autonomousneuroprotective effects on neurons against oxidative insult2728, and the expression of genes encoding the secreted factors thrombospondins 1and 2 (TSP-1 and TSP-2), which are known to promotesynapse formation of neurons29. Interestingly, DS astrogliaexpressed lower levels of NFE2L2, TSP-1 and TSP-2 (Fig. 2a4), indicatingtheir potentially compromised neuroprotective and neurotrophic properties. Notethat variability of neural differentiation of different hiPSC lines wasreported30. We showed that variations were found within theDS astroglia and control astroglia derived from different hiPSCs (Fig. 2a). For example, compared with the control astroglia, DS1astroglia had a prominent increase of S100B gene expression (>74-fold), whereas DS2had a smaller increase (~2.5-fold). To ensure the reliability of ourobservations, each sample was prepared in triplicate and experiments wererepeated for at least three times. Conclusions were drawn only when each DS lineshowed the same trends of change with statistical significance when comparedwith each control line.

Bottom Line: DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules.Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo.Finally, we show that the FDA-approved antibiotic drug, minocycline, partially corrects the pathological phenotypes of DS astroglia by specifically modulating the expression of S100B, GFAP, inducible nitric oxide synthase, and thrombospondins 1 and 2 in DS astroglia.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, California 95817, USA [2] Institute for Pediatric Regenerative Medicine, Shriners Hospitals for Children, Sacramento, California 95817, USA [3] Department of Neurology, Institute of Neurology, Tianjin General Hospital, Tianjin Medical University, Tianjin 300070, China [4].

ABSTRACT
Down's syndrome (DS), caused by trisomy of human chromosome 21, is the most common genetic cause of intellectual disability. Here we use induced pluripotent stem cells (iPSCs) derived from DS patients to identify a role for astrocytes in DS pathogenesis. DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules. Astrocyte-conditioned medium collected from DS astroglia causes toxicity to neurons, and fails to promote neuronal ion channel maturation and synapse formation. Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo. We also observed abnormal gene expression profiles from DS astroglia. Finally, we show that the FDA-approved antibiotic drug, minocycline, partially corrects the pathological phenotypes of DS astroglia by specifically modulating the expression of S100B, GFAP, inducible nitric oxide synthase, and thrombospondins 1 and 2 in DS astroglia. Our studies shed light on the pathogenesis and possible treatment of DS by targeting astrocytes with a clinically available drug.

No MeSH data available.


Related in: MedlinePlus