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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

Transplantation of astroglia into the developing brains of Rag1−/−mice.(a) Representative images showing that at 6 weeks aftertransplantation, the transplanted Di-DS3 Astros and Tri-DS3 Astros wereidentified by human nuclei (hN) staining. Notably, the majority of thetransplanted astroglia (circled by dotted lines) were found integrated intothe tissue and located at the bottom of the LVs. LV, lateral ventricle;DAPI, 4′,6-diamidino-2-phenylindoledihydrochloride. Scale bars, 500 μm.(b) Quantitative results from brain sections showing that nodifference in engraftment success (hN+ cells) was noted between Di-DS3 Astroand Tri-DS3 Astro transplantation groups (n=4–5). NS, nosignificance. (c) A representative image showing that thetransplanted astroglia were labelled by human CD44. Scale bars,50 μm. (d) A representative image showing thatabout 50% of the transplanted hN+ cells were positive for GFAP staining. The squared area ind was enlarged in e, showing the co-localization of hN andGFAP. Scale bars, 50and 25 μm in the original and enlarged images,respectively. (f) Some transplanted astroglia showed longGFAP+ processes, asindicated by arrowheads. The arrow indicates the cell body. Scale bar,50 μm. (g) Representative of DCX and Ki67/nestin staining in dorsal SVZperformed on sections from control (Cont) group received PBS vehicle, groupsreceived Di-DS3 astroglia and Tri-DS3 astroglia transplant, and a groupreceived Tri-DS3 astroglia transplant plus minocycline treatment. Scale bars,50 μm. (h,i) Quantitative analysis of thenumber of DCX+ andKi67+ cells at dorsalSVZ in the different groups. (j) Quantitative analysis offluorescence intensity of the nestin staining. One-way analysis of variance test,n=4–6; *P<0.05, **P<0.01;Data are presented as mean±s.e.m.
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f8: Transplantation of astroglia into the developing brains of Rag1−/−mice.(a) Representative images showing that at 6 weeks aftertransplantation, the transplanted Di-DS3 Astros and Tri-DS3 Astros wereidentified by human nuclei (hN) staining. Notably, the majority of thetransplanted astroglia (circled by dotted lines) were found integrated intothe tissue and located at the bottom of the LVs. LV, lateral ventricle;DAPI, 4′,6-diamidino-2-phenylindoledihydrochloride. Scale bars, 500 μm.(b) Quantitative results from brain sections showing that nodifference in engraftment success (hN+ cells) was noted between Di-DS3 Astroand Tri-DS3 Astro transplantation groups (n=4–5). NS, nosignificance. (c) A representative image showing that thetransplanted astroglia were labelled by human CD44. Scale bars,50 μm. (d) A representative image showing thatabout 50% of the transplanted hN+ cells were positive for GFAP staining. The squared area ind was enlarged in e, showing the co-localization of hN andGFAP. Scale bars, 50and 25 μm in the original and enlarged images,respectively. (f) Some transplanted astroglia showed longGFAP+ processes, asindicated by arrowheads. The arrow indicates the cell body. Scale bar,50 μm. (g) Representative of DCX and Ki67/nestin staining in dorsal SVZperformed on sections from control (Cont) group received PBS vehicle, groupsreceived Di-DS3 astroglia and Tri-DS3 astroglia transplant, and a groupreceived Tri-DS3 astroglia transplant plus minocycline treatment. Scale bars,50 μm. (h,i) Quantitative analysis of thenumber of DCX+ andKi67+ cells at dorsalSVZ in the different groups. (j) Quantitative analysis offluorescence intensity of the nestin staining. One-way analysis of variance test,n=4–6; *P<0.05, **P<0.01;Data are presented as mean±s.e.m.

Mentions: To further examine the pathological phenotypes of DS astroglia in vivo, wetransplanted isogenic Di-DS3 and Tri-DS3 astroglia into the lateral ventricles(LVs) of P0 rag1−/− immunodeficient mouse brains. Sixweeks later, no tumour formation or overgrowth of the transplanted cells wasobserved. As shown in Fig. 8a, both Di-DS3 and Tri-DS3astroglia survived in the mouse brain and were identified by human nuclei (hN)staining. The majority of the transplanted cells were found integrated into thehost tissue and located at the bottom of the LVs. The engraftment efficiency wassimilar between the two astroglia transplantation groups (Fig.8b). Six weeks after transplantation, <1% of the hN+transplanted cells expressed NG2, suggesting that the vast majority of the transplantedcells did not give rise to oligodendroglial lineage cells (Supplementary Fig. 8A). Occasionally, hN+cells were found in the dorsal subventricular zone (SVZ) and vertical medialwall of LV (Supplementary Fig. 8B).But none of them expressed DCX, a marker for immature neurons, indicating that thetransplanted cells did not generate any neurons. These transplanted cellsmaintained their astroglial lineage properties, as indicated by expressing humanCD44 (Fig.8c). Moreover, about half of the transplanted cells expressedGFAP (Fig.8d, 45.6±4.4% and 47.1±5.3% for Di-DS3 astrogliaand Tri-DS astroglia, respectively; n=4–5). Consistent with arecent transplantation study using human glial progenitors isolated from fetalbrain tissues48, many of the transplanted astroglia were large insize and their processes were often long and tortuous (Fig.8e,f), similar to the properties of astroglia in adult human and apebrain6. Next, to explore the effect of DS astroglia onneurogenesis in vivo, we examined the DCX+ cells in the dorsal SVZ, where newly generatedneuroblasts accumulated. Interestingly, as shown in Fig.8g, grafted Di-DS3 astroglia, but not Tri-DS3 astroglia significantlypromoted the endogenous neurogenesis, as indicated by DCX staining (Fig.8h, the numbers of DCX+ cells per section were 116.8±12.7,290.0±27.9 and 141.0±27.0 for control phosphate-bufferedsaline (PBS) vehicle, Di-DS3 astroglia and Tri-DS3 astroglia groups,respectively; P<0.01, n=4–6). Intriguingly,treating the Tri-DS3 astroglia-recipient animals with minocycline promoted neurogenesis inthe dorsal SVZ (Fig. 8g,h, the number of DCX+ cells per section was230.0±25.4; P<0.05 compared with the control vehiclegroup and the group received Tri-DS3 alone, n=4–6). The numberof proliferating cells identified by Ki67 staining in the dorsal SVZ was also significantlyincreased by the transplanted Di-DS3 astroglia compared with the control group(Fig. 8g,i, the cell numbers per section were113.8±9.7 and 160.2±13.9 for control and Di-DS3 astrogliagroups, respectively; P<0.01, n=4–6). However,transplantation of Tri-DS3 astroglia alone or plus minocycline treatment did not changethe number of Ki67+ cells(Fig. 8g,i, the cell numbers per section were91.4±8.7 and 84.7±3.8 for Tri-DS3 astroglia and Tri-DSastroglia plus minocyclinetreatment groups, respectively; P>0.05, n=4–6).As shown in Fig. 8g, we also examined the endogenousneural stem cells labelled by nestin in the dorsal SVZ among different groups. Thedistribution areas of endogenous neural stem cells were expanded in groupsreceived cell transplant, particularly in the group transplanted with Di-DS3astroglia, but quantification of fluorescence intensity of nestin staining did not showsignificant difference among the different groups (Fig.8j; P>0.05, n=4–6). As an additionalcontrol, we also treated the animals that received PBS vehicle withminocycline for 3 weeks.Neither the number of DCX+and Ki67+ cells nor thefluorescence intensity of nestin staining was significantly changed, suggesting thatminocycline exerted itseffects by modulating the transplanted Tri-DS3 astroglia. Although DS ACMincreased neuronal cell death in vitro (Fig. 3b,eand Fig. 7e,h), we did not see any significant neuronalcell death in the animals that received astroglial transplant, as indicated bydouble staining of NeuN and active caspase-3 (Supplementary Fig. 8C).


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)

Transplantation of astroglia into the developing brains of Rag1−/−mice.(a) Representative images showing that at 6 weeks aftertransplantation, the transplanted Di-DS3 Astros and Tri-DS3 Astros wereidentified by human nuclei (hN) staining. Notably, the majority of thetransplanted astroglia (circled by dotted lines) were found integrated intothe tissue and located at the bottom of the LVs. LV, lateral ventricle;DAPI, 4′,6-diamidino-2-phenylindoledihydrochloride. Scale bars, 500 μm.(b) Quantitative results from brain sections showing that nodifference in engraftment success (hN+ cells) was noted between Di-DS3 Astroand Tri-DS3 Astro transplantation groups (n=4–5). NS, nosignificance. (c) A representative image showing that thetransplanted astroglia were labelled by human CD44. Scale bars,50 μm. (d) A representative image showing thatabout 50% of the transplanted hN+ cells were positive for GFAP staining. The squared area ind was enlarged in e, showing the co-localization of hN andGFAP. Scale bars, 50and 25 μm in the original and enlarged images,respectively. (f) Some transplanted astroglia showed longGFAP+ processes, asindicated by arrowheads. The arrow indicates the cell body. Scale bar,50 μm. (g) Representative of DCX and Ki67/nestin staining in dorsal SVZperformed on sections from control (Cont) group received PBS vehicle, groupsreceived Di-DS3 astroglia and Tri-DS3 astroglia transplant, and a groupreceived Tri-DS3 astroglia transplant plus minocycline treatment. Scale bars,50 μm. (h,i) Quantitative analysis of thenumber of DCX+ andKi67+ cells at dorsalSVZ in the different groups. (j) Quantitative analysis offluorescence intensity of the nestin staining. One-way analysis of variance test,n=4–6; *P<0.05, **P<0.01;Data are presented as mean±s.e.m.
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f8: Transplantation of astroglia into the developing brains of Rag1−/−mice.(a) Representative images showing that at 6 weeks aftertransplantation, the transplanted Di-DS3 Astros and Tri-DS3 Astros wereidentified by human nuclei (hN) staining. Notably, the majority of thetransplanted astroglia (circled by dotted lines) were found integrated intothe tissue and located at the bottom of the LVs. LV, lateral ventricle;DAPI, 4′,6-diamidino-2-phenylindoledihydrochloride. Scale bars, 500 μm.(b) Quantitative results from brain sections showing that nodifference in engraftment success (hN+ cells) was noted between Di-DS3 Astroand Tri-DS3 Astro transplantation groups (n=4–5). NS, nosignificance. (c) A representative image showing that thetransplanted astroglia were labelled by human CD44. Scale bars,50 μm. (d) A representative image showing thatabout 50% of the transplanted hN+ cells were positive for GFAP staining. The squared area ind was enlarged in e, showing the co-localization of hN andGFAP. Scale bars, 50and 25 μm in the original and enlarged images,respectively. (f) Some transplanted astroglia showed longGFAP+ processes, asindicated by arrowheads. The arrow indicates the cell body. Scale bar,50 μm. (g) Representative of DCX and Ki67/nestin staining in dorsal SVZperformed on sections from control (Cont) group received PBS vehicle, groupsreceived Di-DS3 astroglia and Tri-DS3 astroglia transplant, and a groupreceived Tri-DS3 astroglia transplant plus minocycline treatment. Scale bars,50 μm. (h,i) Quantitative analysis of thenumber of DCX+ andKi67+ cells at dorsalSVZ in the different groups. (j) Quantitative analysis offluorescence intensity of the nestin staining. One-way analysis of variance test,n=4–6; *P<0.05, **P<0.01;Data are presented as mean±s.e.m.
Mentions: To further examine the pathological phenotypes of DS astroglia in vivo, wetransplanted isogenic Di-DS3 and Tri-DS3 astroglia into the lateral ventricles(LVs) of P0 rag1−/− immunodeficient mouse brains. Sixweeks later, no tumour formation or overgrowth of the transplanted cells wasobserved. As shown in Fig. 8a, both Di-DS3 and Tri-DS3astroglia survived in the mouse brain and were identified by human nuclei (hN)staining. The majority of the transplanted cells were found integrated into thehost tissue and located at the bottom of the LVs. The engraftment efficiency wassimilar between the two astroglia transplantation groups (Fig.8b). Six weeks after transplantation, <1% of the hN+transplanted cells expressed NG2, suggesting that the vast majority of the transplantedcells did not give rise to oligodendroglial lineage cells (Supplementary Fig. 8A). Occasionally, hN+cells were found in the dorsal subventricular zone (SVZ) and vertical medialwall of LV (Supplementary Fig. 8B).But none of them expressed DCX, a marker for immature neurons, indicating that thetransplanted cells did not generate any neurons. These transplanted cellsmaintained their astroglial lineage properties, as indicated by expressing humanCD44 (Fig.8c). Moreover, about half of the transplanted cells expressedGFAP (Fig.8d, 45.6±4.4% and 47.1±5.3% for Di-DS3 astrogliaand Tri-DS astroglia, respectively; n=4–5). Consistent with arecent transplantation study using human glial progenitors isolated from fetalbrain tissues48, many of the transplanted astroglia were large insize and their processes were often long and tortuous (Fig.8e,f), similar to the properties of astroglia in adult human and apebrain6. Next, to explore the effect of DS astroglia onneurogenesis in vivo, we examined the DCX+ cells in the dorsal SVZ, where newly generatedneuroblasts accumulated. Interestingly, as shown in Fig.8g, grafted Di-DS3 astroglia, but not Tri-DS3 astroglia significantlypromoted the endogenous neurogenesis, as indicated by DCX staining (Fig.8h, the numbers of DCX+ cells per section were 116.8±12.7,290.0±27.9 and 141.0±27.0 for control phosphate-bufferedsaline (PBS) vehicle, Di-DS3 astroglia and Tri-DS3 astroglia groups,respectively; P<0.01, n=4–6). Intriguingly,treating the Tri-DS3 astroglia-recipient animals with minocycline promoted neurogenesis inthe dorsal SVZ (Fig. 8g,h, the number of DCX+ cells per section was230.0±25.4; P<0.05 compared with the control vehiclegroup and the group received Tri-DS3 alone, n=4–6). The numberof proliferating cells identified by Ki67 staining in the dorsal SVZ was also significantlyincreased by the transplanted Di-DS3 astroglia compared with the control group(Fig. 8g,i, the cell numbers per section were113.8±9.7 and 160.2±13.9 for control and Di-DS3 astrogliagroups, respectively; P<0.01, n=4–6). However,transplantation of Tri-DS3 astroglia alone or plus minocycline treatment did not changethe number of Ki67+ cells(Fig. 8g,i, the cell numbers per section were91.4±8.7 and 84.7±3.8 for Tri-DS3 astroglia and Tri-DSastroglia plus minocyclinetreatment groups, respectively; P>0.05, n=4–6).As shown in Fig. 8g, we also examined the endogenousneural stem cells labelled by nestin in the dorsal SVZ among different groups. Thedistribution areas of endogenous neural stem cells were expanded in groupsreceived cell transplant, particularly in the group transplanted with Di-DS3astroglia, but quantification of fluorescence intensity of nestin staining did not showsignificant difference among the different groups (Fig.8j; P>0.05, n=4–6). As an additionalcontrol, we also treated the animals that received PBS vehicle withminocycline for 3 weeks.Neither the number of DCX+and Ki67+ cells nor thefluorescence intensity of nestin staining was significantly changed, suggesting thatminocycline exerted itseffects by modulating the transplanted Tri-DS3 astroglia. Although DS ACMincreased neuronal cell death in vitro (Fig. 3b,eand Fig. 7e,h), we did not see any significant neuronalcell death in the animals that received astroglial transplant, as indicated bydouble staining of NeuN and active caspase-3 (Supplementary Fig. 8C).

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