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Cited by 122Down syndrome and intellectual disability researchNeuroscience and Neuropharmacology ResearchGenetics and Neurodevelopmental Disorders

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A Role for Thrombospondin-1 Deficits in Astrocyte-Mediated Spine and Synaptic Pathology in Down's Syndrome

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Garcia, Octavio, Torres, Maria, Helguera, Pablo, Coskun, Pinar, Busciglio, Jorge·2010·PLoS ONE
DOI 10.1371/journal.pone.0014200PMC PMC2996288
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Abstract

BACKGROUND: Down's syndrome (DS) is the most common genetic cause of mental retardation. Reduced number and aberrant architecture of dendritic spines are common features of DS neuropathology. However, the mechanisms involved in DS spine alterations are not known. In addition to a relevant role in synapse formation and maintenance, astrocytes can regulate spine dynamics by releasing soluble factors or by physical contact with neurons. We have previously shown impaired mitochondrial function in DS astrocytes leading to metabolic alterations in protein processing and secretion. In this study, we investigated whether deficits in astrocyte function contribute to DS spine pathology. METHODOLOGY/PRINCIPAL FINDINGS: Using a human astrocyte/rat hippocampal neuron coculture, we found that DS astrocytes are directly involved in the development of spine malformations and reduced synaptic density. We also show that thrombospondin 1 (TSP-1), an astrocyte-secreted protein, possesses a potent modulatory effect on spine number and morphology, and that both DS brains and DS astrocytes exhibit marked deficits in TSP-1 protein expression. Depletion of TSP-1 from normal astrocytes resulted in dramatic changes in spine morphology, while restoration of TSP-1 levels prevented DS astrocyte-mediated spine and synaptic alterations. Astrocyte cultures derived from TSP-1 KO mice exhibited similar deficits to support spine formation and structure than DS astrocytes. CONCLUSIONS/SIGNIFICANCE: These results indicate that human astrocytes promote spine and synapse formation, identify astrocyte dysfunction as a significant factor of spine and synaptic pathology in the DS brain, and provide a mechanistic rationale for the exploration of TSP-1-based therapies to treat spine and synaptic pathology in DS and other neurological conditions.

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Figure 1 Human astrocyte/rat hippocampal neuron cocultures. Cocultures were prepared as described in the Methods section. A) After 21 DIV, the cells were fixed and immunostained with anti-GFAP (1∶1000, red) to visualize astrocytes, and anti-β tubulin class III (1∶1000, green) to visualize neurons. Nuclei were counterstained with Hoechst (blue). Note the significant development of neuronal processes in the cocultures. B) Spines (arrows) present in a typical dendritic segment visualized with anti-drebrin (1∶250, red), and anti-β tubulin class III (blue). Scale bars: A: 20 µm upper panel; 10 µm lower panel. B: 5 µm.
Figure 1 Human astrocyte/rat hippocampal neuron cocultures. Cocultures were prepared as described in the Methods section. A) After 21 DIV, the cells were fixed and immunostained with anti-GFAP (1∶1000, red) to visualize astrocytes, and anti-β tubulin class III (1∶1000, green) to visualize neurons. Nuclei were counterstained with Hoechst (blue). Note the significant development of neuronal processes in the cocultures. B) Spines (arrows) present in a typical dendritic segment visualized with anti-drebrin (1∶250, red), and anti-β tubulin class III (blue). Scale bars: A: 20 µm upper panel; 10 µm lower panel. B: 5 µm.CC BY · PubMed Central
Figure 2 Spine number and morphology are altered in hippocampal neurons grown on top of DS astrocytes. A) Double immunofluorescence with anti-drebrin (red) and anti- β tubulin class III (green) illustrates typical spine morphologies (arrows) found in cocultures of neurons with NL and DS astrocytes respectively. The frequency of filopodium spines is significantly increased in DS cocultures. Scale bar: 5 µm. B) Image analysis indicates a significant reduction in the total number of spines in neurons grown on top of DS astrocytes or maintained in DS CM. In particular, this reduction affects stubby spines (C). Assessment of spine morphology in DS astrocyte cocultures shows a significant increase in the number (D) (NL 3.7±0.5; DS 4.6±0.2 SEM), and length (E) (NL 4.4±0.5; DS 7.5±1 SEM) of filopodium spines. Spine number represents the average number of spines scored in a 50 µm dendritic segment. Data were analyzed by one-way analysis of variance (ANOVA) followed by Fisher's test. All data are expressed as mean ± SEM. *p<0.05.
Figure 2 Spine number and morphology are altered in hippocampal neurons grown on top of DS astrocytes. A) Double immunofluorescence with anti-drebrin (red) and anti- β tubulin class III (green) illustrates typical spine morphologies (arrows) found in cocultures of neurons with NL and DS astrocytes respectively. The frequency of filopodium spines is significantly increased in DS cocultures. Scale bar: 5 µm. B) Image analysis indicates a significant reduction in the total number of spines in neurons grown on top of DS astrocytes or maintained in DS CM. In particular, this reduction affects stubby spines (C). Assessment of spine morphology in DS astrocyte cocultures shows a significant increase in the number (D) (NL 3.7±0.5; DS 4.6±0.2 SEM), and length (E) (NL 4.4±0.5; DS 7.5±1 SEM) of filopodium spines. Spine number represents the average number of spines scored in a 50 µm dendritic segment. Data were analyzed by one-way analysis of variance (ANOVA) followed by Fisher's test. All data are expressed as mean ± SEM. *p<0.05.CC BY · PubMed Central
Figure 3 Reduced TSP-1 levels in DS astrocytes and DS fetal brain tissue. Human astocytes in culture express abundant TSP-1. A) Differential interference contrast microscopy (DIC) image of the field shown in B-D. B) Cell surface TSP-1 IF prior to permeabilization of the cells (red fluorescence). C) After permeabilization, the preparation was incubated again with anti-TSP-1 (green fluorescence). Note the strong perinuclear staining. D) Merged image of the fields shown in B and C. Nuclei were counterstained with Hoechst (blue). Scale bar: 10 µm. E) Measurement of TSP-1 levels in homogenates (cellular) and CM (secreted) indicates significant reductions in DS astrocytes. Homogenates: NL 88.4±11.1 ng/ml, DS 51.51±4.7 ng/ml; CM: NL 564.0±54.2 ng/ml, DS 381.9±42.0 ng/ml. F) TSP-1 levels were measured in NL (n = 6) and DS (n = 6) fetal cortical brain samples as described in the Methods section. Average gestational ages of NL and DS samples were 22.3 weeks and 18.5 weeks respectively. The concentration of TSP-1 was markedly reduced in DS brain samples (average values above of the bars). Data were analyzed by ANOVA followed by Fisher's test. Error bars indicate the mean ± SEM. *p<0.05.
Figure 3 Reduced TSP-1 levels in DS astrocytes and DS fetal brain tissue. Human astocytes in culture express abundant TSP-1. A) Differential interference contrast microscopy (DIC) image of the field shown in B-D. B) Cell surface TSP-1 IF prior to permeabilization of the cells (red fluorescence). C) After permeabilization, the preparation was incubated again with anti-TSP-1 (green fluorescence). Note the strong perinuclear staining. D) Merged image of the fields shown in B and C. Nuclei were counterstained with Hoechst (blue). Scale bar: 10 µm. E) Measurement of TSP-1 levels in homogenates (cellular) and CM (secreted) indicates significant reductions in DS astrocytes. Homogenates: NL 88.4±11.1 ng/ml, DS 51.51±4.7 ng/ml; CM: NL 564.0±54.2 ng/ml, DS 381.9±42.0 ng/ml. F) TSP-1 levels were measured in NL (n = 6) and DS (n = 6) fetal cortical brain samples as described in the Methods section. Average gestational ages of NL and DS samples were 22.3 weeks and 18.5 weeks respectively. The concentration of TSP-1 was markedly reduced in DS brain samples (average values above of the bars). Data were analyzed by ANOVA followed by Fisher's test. Error bars indicate the mean ± SEM. *p<0.05.CC BY · PubMed Central
Figure 4 TSP-1 modulates spine development. A) Pure rat hippocampal cultures were incubated with BDNF (10 ng/ml), recombinant TSP-1 (250 ng/ml), or TSP-1+BDNF as described in the Methods section. Double immunofluorescence shows anti-Drebrin (red) and anti-β tubulin class III (green) immunostaining. Note abundant spine development with the treatments. Nuclei were counterstained with Hoechst (blue). Scale bar: 10 µm. B) TSP-1 and BDNF significantly enhance total spine number to similar levels. Coincubation with TSP-1+ BDNF produces a higher increase in spine number than either individual factor. Error bars indicate the mean ± SEM. *p<0.05 vs Control, **p<0.05 vs TSP-1 or BDNF. C) Increasing concentrations of TSP-1 induces a gradual increase in spine density in hippocampal neurons. Error bars indicate the mean ± SEM. *p<0.05.
Figure 4 TSP-1 modulates spine development. A) Pure rat hippocampal cultures were incubated with BDNF (10 ng/ml), recombinant TSP-1 (250 ng/ml), or TSP-1+BDNF as described in the Methods section. Double immunofluorescence shows anti-Drebrin (red) and anti-β tubulin class III (green) immunostaining. Note abundant spine development with the treatments. Nuclei were counterstained with Hoechst (blue). Scale bar: 10 µm. B) TSP-1 and BDNF significantly enhance total spine number to similar levels. Coincubation with TSP-1+ BDNF produces a higher increase in spine number than either individual factor. Error bars indicate the mean ± SEM. *p<0.05 vs Control, **p<0.05 vs TSP-1 or BDNF. C) Increasing concentrations of TSP-1 induces a gradual increase in spine density in hippocampal neurons. Error bars indicate the mean ± SEM. *p<0.05.CC BY · PubMed Central
Figure 5 Depletion of TSP-1 markedly alters spine morphology. Cocultures of rat hippocampal neurons and NL astrocytes were incubated with a control IgG (A) or anti- TSP-1 (B). The region outlined by rectangles is shown at higher magnification in the panels below. Cocultures immunodepleted of TSP-1 exhibit dramatic changes in spine morphology including a marked increase in long filopodium-like spines (arrows) compared to control cultures, in which stubby spines predominate (arrows). Cocultures were fixed and immunofluorescence was performed with anti-drebrin (red) and anti- β tubulin class III (green) antibodies. The panels at higher magnification have been pseudocolored in red (tubulin) and white (drebrin) to facilitate the visualization of spine morphologies. Nuclei were counterstained with Hoechst (blue). Scale bars: 10 µm (upper panel), 5 µm (lower panel).
Figure 5 Depletion of TSP-1 markedly alters spine morphology. Cocultures of rat hippocampal neurons and NL astrocytes were incubated with a control IgG (A) or anti- TSP-1 (B). The region outlined by rectangles is shown at higher magnification in the panels below. Cocultures immunodepleted of TSP-1 exhibit dramatic changes in spine morphology including a marked increase in long filopodium-like spines (arrows) compared to control cultures, in which stubby spines predominate (arrows). Cocultures were fixed and immunofluorescence was performed with anti-drebrin (red) and anti- β tubulin class III (green) antibodies. The panels at higher magnification have been pseudocolored in red (tubulin) and white (drebrin) to facilitate the visualization of spine morphologies. Nuclei were counterstained with Hoechst (blue). Scale bars: 10 µm (upper panel), 5 µm (lower panel).CC BY · PubMed Central
Figure 6 TSP-1 prevents spine alterations in neurons grown on top of DS astrocytes. Recombinant TSP-1 (250 ng/ml) was added to DS astrocyte/hippocampal neuron cocultures. TSP-1 induced a significant reduction in the number of filopodium-like spines (DS: 5.3±0.5; DS+TSP-1: 1.2±0.1)(A), and a marked increase in the number of stubby spines (DS: 3.5±0.3; DS+TSP-1: 7.2±0.5)(B). Heat-inactivated (H.I.) TSP-1 did not have any effect on spines. Data were analyzed by ANOVA followed by Fisher's test. Error bars indicate the mean ± SEM. *p<0.05, **p<0.05.
Figure 6 TSP-1 prevents spine alterations in neurons grown on top of DS astrocytes. Recombinant TSP-1 (250 ng/ml) was added to DS astrocyte/hippocampal neuron cocultures. TSP-1 induced a significant reduction in the number of filopodium-like spines (DS: 5.3±0.5; DS+TSP-1: 1.2±0.1)(A), and a marked increase in the number of stubby spines (DS: 3.5±0.3; DS+TSP-1: 7.2±0.5)(B). Heat-inactivated (H.I.) TSP-1 did not have any effect on spines. Data were analyzed by ANOVA followed by Fisher's test. Error bars indicate the mean ± SEM. *p<0.05, **p<0.05.CC BY · PubMed Central
Figure 7 Altered spine morphology in neurons growing on top of TSP-1 KO astrocytes. Rat hippocampal neuron/mouse astrocyte cocultures were prepared as described in the Methods section. (A) After 21 DIV, the cultures were fixed and immunostained with anti-GFAP (1∶1000, red), an astrocytic marker, and anti-β tubulin class III (1∶1000, green), a neuronal marker. Hoechst was used for nuclear staining (blue). Similar neuronal viability and growth was observed in WT and TSP-1 KO (TSP-1−/−) cocultures. Scale bar: 20 µm. (B) Double immunofluorescence with anti-drebrin (red) and anti- β tubulin class III (green) illustrates the differences in spine morphology in neurons growing on top of WT or TSP-1 astrocytes. Note the presence of numerous filopodium spines in TSP-1 KO cocultures. Scale bar: 5 µm. (C) Assessment of spine morphology indicates a significant increase in filopodium spines in TSP-1 KO cocultures (KO) compared to WT cocultures. Conversely, the number of stubby spines is reduced more than 50% in TSP-1 KO cocultures. Continuous treatment with recombinant TSP-1 (250 ng/ml) during 4 days (4D) or 7 days (7D) prior to fixation at day 21 averted the spine alterations in TSP-1 KO cocultures. Spine number represents the average number of spines scored in a 50 µm dendritic segment. Data were analyzed by ANOVA followed by Fisher's test. All data are expressed as mean ± SEM. *p<0.05.
Figure 7 Altered spine morphology in neurons growing on top of TSP-1 KO astrocytes. Rat hippocampal neuron/mouse astrocyte cocultures were prepared as described in the Methods section. (A) After 21 DIV, the cultures were fixed and immunostained with anti-GFAP (1∶1000, red), an astrocytic marker, and anti-β tubulin class III (1∶1000, green), a neuronal marker. Hoechst was used for nuclear staining (blue). Similar neuronal viability and growth was observed in WT and TSP-1 KO (TSP-1−/−) cocultures. Scale bar: 20 µm. (B) Double immunofluorescence with anti-drebrin (red) and anti- β tubulin class III (green) illustrates the differences in spine morphology in neurons growing on top of WT or TSP-1 astrocytes. Note the presence of numerous filopodium spines in TSP-1 KO cocultures. Scale bar: 5 µm. (C) Assessment of spine morphology indicates a significant increase in filopodium spines in TSP-1 KO cocultures (KO) compared to WT cocultures. Conversely, the number of stubby spines is reduced more than 50% in TSP-1 KO cocultures. Continuous treatment with recombinant TSP-1 (250 ng/ml) during 4 days (4D) or 7 days (7D) prior to fixation at day 21 averted the spine alterations in TSP-1 KO cocultures. Spine number represents the average number of spines scored in a 50 µm dendritic segment. Data were analyzed by ANOVA followed by Fisher's test. All data are expressed as mean ± SEM. *p<0.05.CC BY · PubMed Central
Figure 8 Reduced synaptic density in hippocampal neurons grown on top of DS astrocytes can be reversed by treatment with TSP-1. A) Colocalization of post-synaptic marker PSD-95 (1∶250, red) and pre-synaptic marker synaptophysin (1∶250, green)(arrows) was utilized to define synaptic contacts and to assess synaptic density. Neuronal processes were stained with tubulin class III (blue). B) Synaptic density was expressed as the number of colocalized puncta per 50 µm of dendrite in NL, DS and DS treated with 250 ng/ml TSP-1 cocultures. Error bars indicate the mean ± SEM. *p<0.05 vs cocultures of NL astrocytes, **p<0.05 vs cocultures of DS astrocytes. C) The colocalization of synaptophysin, drebrin and tubulin class III denotes the presence of synapses at spines (arrows). D) The number of synapses localized at spines was assessed by quantifying the triple colocalization of synaptophysin, PSD95 and drebrin (arrows). Error bars indicate the mean ± SEM. *p<0.05. E) The fluorescent cationic styryl fixable dye AM4-64, which is taken up during vesicle recycling, was used to study synaptic activity in the cocultures. AM4-64 fluorescence is shown in living NL cultures after depolarization induced by treatment with KCl (upper panel). DS cocultures present a marked decrease in AM4-64-positive vesicles after stimulation with KCl (middle panel). Treatment with TSP-1 increases synaptic activity in DS cocultures (lower panel). F) Quantification of AM4-64-positive puncta shows a significant reduction in DS compared to NL cocultures, which can be reverted by TSP-1 treatment. Data were analyzed by ANOVA followed by Fisher's test. All data are expressed as mean ± SEM. *p<0.05. Scale bars: 5 µm.
Figure 8 Reduced synaptic density in hippocampal neurons grown on top of DS astrocytes can be reversed by treatment with TSP-1. A) Colocalization of post-synaptic marker PSD-95 (1∶250, red) and pre-synaptic marker synaptophysin (1∶250, green)(arrows) was utilized to define synaptic contacts and to assess synaptic density. Neuronal processes were stained with tubulin class III (blue). B) Synaptic density was expressed as the number of colocalized puncta per 50 µm of dendrite in NL, DS and DS treated with 250 ng/ml TSP-1 cocultures. Error bars indicate the mean ± SEM. *p<0.05 vs cocultures of NL astrocytes, **p<0.05 vs cocultures of DS astrocytes. C) The colocalization of synaptophysin, drebrin and tubulin class III denotes the presence of synapses at spines (arrows). D) The number of synapses localized at spines was assessed by quantifying the triple colocalization of synaptophysin, PSD95 and drebrin (arrows). Error bars indicate the mean ± SEM. *p<0.05. E) The fluorescent cationic styryl fixable dye AM4-64, which is taken up during vesicle recycling, was used to study synaptic activity in the cocultures. AM4-64 fluorescence is shown in living NL cultures after depolarization induced by treatment with KCl (upper panel). DS cocultures present a marked decrease in AM4-64-positive vesicles after stimulation with KCl (middle panel). Treatment with TSP-1 increases synaptic activity in DS cocultures (lower panel). F) Quantification of AM4-64-positive puncta shows a significant reduction in DS compared to NL cocultures, which can be reverted by TSP-1 treatment. Data were analyzed by ANOVA followed by Fisher's test. All data are expressed as mean ± SEM. *p<0.05. Scale bars: 5 µm.CC BY · PubMed Central

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Authors
Garcia, O., Torres, M., Helguera, P., Coskun, P., & Busciglio, J.
Affiliations
University of California, Irvine (US)
Corresponding author
Busciglio, Jorge
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2010-12-02
Volume
5
Issue
12
Pages
e14200
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0014200
PMC
PMC2996288
OpenAlex
W2036755685
Cited by
122
References
115
Citations by year
2026: 5 · 2025: 8 · 2024: 9 · 2023: 9 · 2022: 6 · 2021: 7 · 2020: 9 · 2019: 7 · 2018: 7 · 2017: 7 · 2016: 12 · 2015: 4 · 2014: 9 · 2013: 8 · 2012: 12
FWCI
7.32
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • Larry L. Hillblom Foundation
  • National Institutes of Health (AG16573)
  • National Institute on Aging (AG 16573, P50AG016573)
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (HD-38466, R01HD038466)
Topics
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Down syndrome and intellectual disability research
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
Keywords
Down syndrome, thrombospondin-1, astrocytes, dendritic spines, synaptic pathology, dendritic spine morphology, synaptic density, astrocyte-neuron co-culture, hippocampal neurons, intellectual disability, astrocyte dysfunction
MeSH terms
Animals, Astrocytes, Brain, Down Syndrome, Humans, Microscopy, Fluorescence, Neurons, Synapses, Gene Expression Regulation, Developmental, Coculture Techniques, Thrombospondin 1, Animals, Genetically Modified, Dendritic Spines, Rats

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