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Systemic attenuation of the TGF-β pathway by a single drug simultaneously rejuvenates hippocampal neurogenesis and myogenesis in the same old mammal.

Yousef H, Conboy MJ, Morgenthaler A, Schlesinger C, Bugaj L, Paliwal P, Greer C, Conboy IM, Schaffer D - Oncotarget (2015)

Bottom Line: At the levels of cellular mechanism, our results establish that the age-specific increase in TGF-β1 in the stem cell niches of aged hippocampus involves microglia and that such an increase is pro-inflammatory both in brain and muscle, as assayed by the elevated expression of β2 microglobulin (B2M), a component of MHC class I molecules.These findings suggest that at high levels typical of aged tissues, TGF-β1 promotes inflammation instead of its canonical role in attenuating immune responses.In agreement with this conclusion, inhibition of TGF-β1 signaling normalized B2M to young levels in both studied tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, UC Berkeley, Berkeley, CA, USA.

ABSTRACT
Stem cell function declines with age largely due to the biochemical imbalances in their tissue niches, and this work demonstrates that aging imposes an elevation in transforming growth factor β (TGF-β) signaling in the neurogenic niche of the hippocampus, analogous to the previously demonstrated changes in the myogenic niche of skeletal muscle with age. Exploring the hypothesis that youthful calibration of key signaling pathways may enhance regeneration of multiple old tissues, we found that systemically attenuating TGF-β signaling with a single drug simultaneously enhanced neurogenesis and muscle regeneration in the same old mice, findings further substantiated via genetic perturbations. At the levels of cellular mechanism, our results establish that the age-specific increase in TGF-β1 in the stem cell niches of aged hippocampus involves microglia and that such an increase is pro-inflammatory both in brain and muscle, as assayed by the elevated expression of β2 microglobulin (B2M), a component of MHC class I molecules. These findings suggest that at high levels typical of aged tissues, TGF-β1 promotes inflammation instead of its canonical role in attenuating immune responses. In agreement with this conclusion, inhibition of TGF-β1 signaling normalized B2M to young levels in both studied tissues.

No MeSH data available.


Related in: MedlinePlus

Downstream effectors of TGF-β signaling increase with age in mice hippocampi and inhibits neural progenitor cell proliferationA. Immunofluorescence staining for Sox2 (green) and pSmad3 (red) was performed on young and old brain tissue sections (n = 4 young, 6 old), with DAPI (blue) labeling all nuclei. Representative images are shown. Scale bar = 50 μM. B. MetaXpress image quantification was developed to calculate the average pixel intensity specifically in Sox2+ neural stem and progenitor cells in the SGZ of the dentate gyrus. Significant differences were identified by Student's t-tests (two-tailed) (*p < 0.001). Error bars indicate standard error of the mean (n = 4 young, 6 old). C. qRT-PCR quantification of p21 mRNA expression from young and old hippocampi. The relative average expression level was normalized by GAPDH and presented relative to young hippocampi. Significant differences were identified by Student's t-tests (two-tailed) (*p < 0.003), and error bars indicate standard error of the mean (n = 3 young, 3 old biological replicates per group). D. Immunoblotting analysis of a downstream effector of TGF-β, pSmad3, from NPCs cultured in growth medium in the presence or absence of TGF-β1 (50 ng/mL) for 30 minutes. pSmad3 signaling is induced through TGF-β1, as compared with levels of total SMAD2/3. E. Functional validation of increasing TGF-β1. NPCs were cultured in growth medium (DMF12 + N2 + 10 ng/mL FGF-2) in the presence or absence of TGF-β1 (100 ng/mL) for 24 hrs. A 2 hour BrdU (10 μM) pulse was performed before cell fixation to label proliferating cells. Staining was conducted for BrdU (green) and Sox2 (red), with Hoechst (blue) labeling all nuclei. Representative images are shown. Scale bar = 100 μM. F. Proliferation of NPCs was quantified by cell scoring in 25 random fields of each condition using an automated imager and MetaXpress cell scoring software. Results are displayed as the mean percent of BrdU+ proliferating NPCs +/−SD, respectively. Significant differences were identified by Student's t-tests (two-tailed) (*p < 0.0003), and error bars indicate standard error of the mean (n = 4 biological replicates).
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Figure 3: Downstream effectors of TGF-β signaling increase with age in mice hippocampi and inhibits neural progenitor cell proliferationA. Immunofluorescence staining for Sox2 (green) and pSmad3 (red) was performed on young and old brain tissue sections (n = 4 young, 6 old), with DAPI (blue) labeling all nuclei. Representative images are shown. Scale bar = 50 μM. B. MetaXpress image quantification was developed to calculate the average pixel intensity specifically in Sox2+ neural stem and progenitor cells in the SGZ of the dentate gyrus. Significant differences were identified by Student's t-tests (two-tailed) (*p < 0.001). Error bars indicate standard error of the mean (n = 4 young, 6 old). C. qRT-PCR quantification of p21 mRNA expression from young and old hippocampi. The relative average expression level was normalized by GAPDH and presented relative to young hippocampi. Significant differences were identified by Student's t-tests (two-tailed) (*p < 0.003), and error bars indicate standard error of the mean (n = 3 young, 3 old biological replicates per group). D. Immunoblotting analysis of a downstream effector of TGF-β, pSmad3, from NPCs cultured in growth medium in the presence or absence of TGF-β1 (50 ng/mL) for 30 minutes. pSmad3 signaling is induced through TGF-β1, as compared with levels of total SMAD2/3. E. Functional validation of increasing TGF-β1. NPCs were cultured in growth medium (DMF12 + N2 + 10 ng/mL FGF-2) in the presence or absence of TGF-β1 (100 ng/mL) for 24 hrs. A 2 hour BrdU (10 μM) pulse was performed before cell fixation to label proliferating cells. Staining was conducted for BrdU (green) and Sox2 (red), with Hoechst (blue) labeling all nuclei. Representative images are shown. Scale bar = 100 μM. F. Proliferation of NPCs was quantified by cell scoring in 25 random fields of each condition using an automated imager and MetaXpress cell scoring software. Results are displayed as the mean percent of BrdU+ proliferating NPCs +/−SD, respectively. Significant differences were identified by Student's t-tests (two-tailed) (*p < 0.0003), and error bars indicate standard error of the mean (n = 4 biological replicates).

Mentions: To confirm and build upon these results, we analyzed downstream pSmad signaling in young versus aged neural stem cells in vivo. As shown in Figure 3A (and quantified in 3B), the levels of pSmad3 were up-regulated in aged compared to young Sox2+ neural stem and progenitor cells in the SGZ. Furthermore, TGF-β1 is a known inhibitor of cell proliferation [26, 27, 35, 37], and accordingly qRT-PCR revealed that in vivo hippocampal expression of p21, a known down-stream target of TGF-β1/pSmad pathway that inhibits cell cycle progression [37, 42], was elevated with age (Figure 3C). To probe whether the age-associated increase in TGF-β expression may be functionally important for neurogenesis, TGF-β1 ligand was added to hippocampal-derived Sox2+ neural progenitor cell (NPCs) cultures and observed to both upregulate pSmad3 (Figure 3D) and decrease cell proliferation, as assayed by reduced BrdU uptake (Figure 3E-3F). These data are the first to profile the age-specific expression of TGF-β family ligands in the neurogenic and myogenic tissue niches. Furthermore, these data are the first to demonstrate that the levels of TGF-β1 transcript and protein and p21 transcript increase with age in the hippocampus and particularly in microglia, and that SMAD3 phosphorylation increases in resident Sox2+ neural stem and progenitor cells of the old hippocampus.


Systemic attenuation of the TGF-β pathway by a single drug simultaneously rejuvenates hippocampal neurogenesis and myogenesis in the same old mammal.

Yousef H, Conboy MJ, Morgenthaler A, Schlesinger C, Bugaj L, Paliwal P, Greer C, Conboy IM, Schaffer D - Oncotarget (2015)

Downstream effectors of TGF-β signaling increase with age in mice hippocampi and inhibits neural progenitor cell proliferationA. Immunofluorescence staining for Sox2 (green) and pSmad3 (red) was performed on young and old brain tissue sections (n = 4 young, 6 old), with DAPI (blue) labeling all nuclei. Representative images are shown. Scale bar = 50 μM. B. MetaXpress image quantification was developed to calculate the average pixel intensity specifically in Sox2+ neural stem and progenitor cells in the SGZ of the dentate gyrus. Significant differences were identified by Student's t-tests (two-tailed) (*p < 0.001). Error bars indicate standard error of the mean (n = 4 young, 6 old). C. qRT-PCR quantification of p21 mRNA expression from young and old hippocampi. The relative average expression level was normalized by GAPDH and presented relative to young hippocampi. Significant differences were identified by Student's t-tests (two-tailed) (*p < 0.003), and error bars indicate standard error of the mean (n = 3 young, 3 old biological replicates per group). D. Immunoblotting analysis of a downstream effector of TGF-β, pSmad3, from NPCs cultured in growth medium in the presence or absence of TGF-β1 (50 ng/mL) for 30 minutes. pSmad3 signaling is induced through TGF-β1, as compared with levels of total SMAD2/3. E. Functional validation of increasing TGF-β1. NPCs were cultured in growth medium (DMF12 + N2 + 10 ng/mL FGF-2) in the presence or absence of TGF-β1 (100 ng/mL) for 24 hrs. A 2 hour BrdU (10 μM) pulse was performed before cell fixation to label proliferating cells. Staining was conducted for BrdU (green) and Sox2 (red), with Hoechst (blue) labeling all nuclei. Representative images are shown. Scale bar = 100 μM. F. Proliferation of NPCs was quantified by cell scoring in 25 random fields of each condition using an automated imager and MetaXpress cell scoring software. Results are displayed as the mean percent of BrdU+ proliferating NPCs +/−SD, respectively. Significant differences were identified by Student's t-tests (two-tailed) (*p < 0.0003), and error bars indicate standard error of the mean (n = 4 biological replicates).
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Figure 3: Downstream effectors of TGF-β signaling increase with age in mice hippocampi and inhibits neural progenitor cell proliferationA. Immunofluorescence staining for Sox2 (green) and pSmad3 (red) was performed on young and old brain tissue sections (n = 4 young, 6 old), with DAPI (blue) labeling all nuclei. Representative images are shown. Scale bar = 50 μM. B. MetaXpress image quantification was developed to calculate the average pixel intensity specifically in Sox2+ neural stem and progenitor cells in the SGZ of the dentate gyrus. Significant differences were identified by Student's t-tests (two-tailed) (*p < 0.001). Error bars indicate standard error of the mean (n = 4 young, 6 old). C. qRT-PCR quantification of p21 mRNA expression from young and old hippocampi. The relative average expression level was normalized by GAPDH and presented relative to young hippocampi. Significant differences were identified by Student's t-tests (two-tailed) (*p < 0.003), and error bars indicate standard error of the mean (n = 3 young, 3 old biological replicates per group). D. Immunoblotting analysis of a downstream effector of TGF-β, pSmad3, from NPCs cultured in growth medium in the presence or absence of TGF-β1 (50 ng/mL) for 30 minutes. pSmad3 signaling is induced through TGF-β1, as compared with levels of total SMAD2/3. E. Functional validation of increasing TGF-β1. NPCs were cultured in growth medium (DMF12 + N2 + 10 ng/mL FGF-2) in the presence or absence of TGF-β1 (100 ng/mL) for 24 hrs. A 2 hour BrdU (10 μM) pulse was performed before cell fixation to label proliferating cells. Staining was conducted for BrdU (green) and Sox2 (red), with Hoechst (blue) labeling all nuclei. Representative images are shown. Scale bar = 100 μM. F. Proliferation of NPCs was quantified by cell scoring in 25 random fields of each condition using an automated imager and MetaXpress cell scoring software. Results are displayed as the mean percent of BrdU+ proliferating NPCs +/−SD, respectively. Significant differences were identified by Student's t-tests (two-tailed) (*p < 0.0003), and error bars indicate standard error of the mean (n = 4 biological replicates).
Mentions: To confirm and build upon these results, we analyzed downstream pSmad signaling in young versus aged neural stem cells in vivo. As shown in Figure 3A (and quantified in 3B), the levels of pSmad3 were up-regulated in aged compared to young Sox2+ neural stem and progenitor cells in the SGZ. Furthermore, TGF-β1 is a known inhibitor of cell proliferation [26, 27, 35, 37], and accordingly qRT-PCR revealed that in vivo hippocampal expression of p21, a known down-stream target of TGF-β1/pSmad pathway that inhibits cell cycle progression [37, 42], was elevated with age (Figure 3C). To probe whether the age-associated increase in TGF-β expression may be functionally important for neurogenesis, TGF-β1 ligand was added to hippocampal-derived Sox2+ neural progenitor cell (NPCs) cultures and observed to both upregulate pSmad3 (Figure 3D) and decrease cell proliferation, as assayed by reduced BrdU uptake (Figure 3E-3F). These data are the first to profile the age-specific expression of TGF-β family ligands in the neurogenic and myogenic tissue niches. Furthermore, these data are the first to demonstrate that the levels of TGF-β1 transcript and protein and p21 transcript increase with age in the hippocampus and particularly in microglia, and that SMAD3 phosphorylation increases in resident Sox2+ neural stem and progenitor cells of the old hippocampus.

Bottom Line: At the levels of cellular mechanism, our results establish that the age-specific increase in TGF-β1 in the stem cell niches of aged hippocampus involves microglia and that such an increase is pro-inflammatory both in brain and muscle, as assayed by the elevated expression of β2 microglobulin (B2M), a component of MHC class I molecules.These findings suggest that at high levels typical of aged tissues, TGF-β1 promotes inflammation instead of its canonical role in attenuating immune responses.In agreement with this conclusion, inhibition of TGF-β1 signaling normalized B2M to young levels in both studied tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, UC Berkeley, Berkeley, CA, USA.

ABSTRACT
Stem cell function declines with age largely due to the biochemical imbalances in their tissue niches, and this work demonstrates that aging imposes an elevation in transforming growth factor β (TGF-β) signaling in the neurogenic niche of the hippocampus, analogous to the previously demonstrated changes in the myogenic niche of skeletal muscle with age. Exploring the hypothesis that youthful calibration of key signaling pathways may enhance regeneration of multiple old tissues, we found that systemically attenuating TGF-β signaling with a single drug simultaneously enhanced neurogenesis and muscle regeneration in the same old mice, findings further substantiated via genetic perturbations. At the levels of cellular mechanism, our results establish that the age-specific increase in TGF-β1 in the stem cell niches of aged hippocampus involves microglia and that such an increase is pro-inflammatory both in brain and muscle, as assayed by the elevated expression of β2 microglobulin (B2M), a component of MHC class I molecules. These findings suggest that at high levels typical of aged tissues, TGF-β1 promotes inflammation instead of its canonical role in attenuating immune responses. In agreement with this conclusion, inhibition of TGF-β1 signaling normalized B2M to young levels in both studied tissues.

No MeSH data available.


Related in: MedlinePlus