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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

B2M levels decrease in neurogenic and myogenic niches following systemic or local attenuation of TGF-β signalingA. qRT-PCR quantification of B2m 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 < 0.04), and error bars indicate standard error of the mean (n = 4 old, 4 young). B. Brain sections spanning the hippocampus from young mice (2 month), as well as Alk5 inhibitor or vehicle treated old (24 month) mice, were immunostained for B2M (red), with Hoechst (blue) labeling all cell nuclei. Representative images are shown. Scale bar = 50 μM. C. Average B2M pixel intensity was quantified using MetaXpress software, and significant differences were identified by Student's t-tests (two-tailed) (*p < 3 × 10−6, **p < 0.0001). Error bars indicate standard error of the mean (n = 5 young, 3 old + Alk5i, 3 old + vehicle biological replicates (mice), with n = 12 technical replicates per mouse) D. Tibialis anterior muscle collected 5 days post-injury were immunostained for B2M (red), with Hoechst (blue) labeling all cell nuclei. Representative images are shown. Scale bar = 100 μM. E. Average B2M pixel intensity of 10 μM tibialis anterior muscle sections throughout sites of injury were quantified using MetaXpress software. Significant differences were identified by Student's t-tests (two-tailed) (*p < 7 × 10−7, **p < 5 × 10−5), and error bars indicate standard error of the mean (n = 3 biological replicates per group (mice), with n = 12 technical replicates per mouse) F. TA muscles collected 5 days post injury were immunostained for B2M (red), with Hoechst (blue) labeling all cell nuclei. Representative images are shown. Scale bar = 100 μM. G. Average B2M pixel intensity of TA muscle sections throughout sites of injury were quantified using MetaXpress software. Significant differences were identified by Student's t-tests (two-tailed) (*p < 0.002). Error bars indicate standard error of the mean (n = 3 biological replicates per group (mice), with n = 12 technical replicates per mouse).
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Figure 7: B2M levels decrease in neurogenic and myogenic niches following systemic or local attenuation of TGF-β signalingA. qRT-PCR quantification of B2m 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 < 0.04), and error bars indicate standard error of the mean (n = 4 old, 4 young). B. Brain sections spanning the hippocampus from young mice (2 month), as well as Alk5 inhibitor or vehicle treated old (24 month) mice, were immunostained for B2M (red), with Hoechst (blue) labeling all cell nuclei. Representative images are shown. Scale bar = 50 μM. C. Average B2M pixel intensity was quantified using MetaXpress software, and significant differences were identified by Student's t-tests (two-tailed) (*p < 3 × 10−6, **p < 0.0001). Error bars indicate standard error of the mean (n = 5 young, 3 old + Alk5i, 3 old + vehicle biological replicates (mice), with n = 12 technical replicates per mouse) D. Tibialis anterior muscle collected 5 days post-injury were immunostained for B2M (red), with Hoechst (blue) labeling all cell nuclei. Representative images are shown. Scale bar = 100 μM. E. Average B2M pixel intensity of 10 μM tibialis anterior muscle sections throughout sites of injury were quantified using MetaXpress software. Significant differences were identified by Student's t-tests (two-tailed) (*p < 7 × 10−7, **p < 5 × 10−5), and error bars indicate standard error of the mean (n = 3 biological replicates per group (mice), with n = 12 technical replicates per mouse) F. TA muscles collected 5 days post injury were immunostained for B2M (red), with Hoechst (blue) labeling all cell nuclei. Representative images are shown. Scale bar = 100 μM. G. Average B2M pixel intensity of TA muscle sections throughout sites of injury were quantified using MetaXpress software. Significant differences were identified by Student's t-tests (two-tailed) (*p < 0.002). Error bars indicate standard error of the mean (n = 3 biological replicates per group (mice), with n = 12 technical replicates per mouse).

Mentions: We compared the B2M expression levels as an indicator of inflammatory response in young and old mice treated with vehicle control, and old mice treated with the Alk5 inhibitor (Figure 4). B2M levels were extremely low in young brain and muscle, consistent with prior work [30], but increased significantly with aging (Figure 7A-7E). Importantly, when TGF-β signaling was attenuated in vivo by the Alk5 inhibitor, B2M levels were significantly diminished both in old brain (Figure 7B, 7C) and old muscle (Figure 7D, 7E). These results demonstrate that B2M becomes upregulated with aging in multiple tissues (suggesting an increase in inflammation) and that down-modulation of TGF-β signaling, which rejuvenates myogenesis and neurogenesis, normalizes B2M in myogenic and neurogenic regions to their young levels, suggesting attenuation of inflammation. As further support of this conclusion, the levels of B2M were also significantly reduced in regenerating regions of the old muscle administered with the dnTGFBR2, as compared to tissue administered with control GFP virus (Figure 7F, 7G). Furthermore, addition of low concentrations of TGF-β1 (0–5 ng/mL) to immune cells, specifically BV2 cells – a microglia cell line – did not affect B2M levels, as assayed by pixel intensity and percent area staining of B2M (Supplemental Figure 6A-6C). In contrast, high TGF-β1 (50 ng/mL) levels induced a significant increase in B2M expression. Thus, physiologically young levels TGF-β1 does not induce B2M, but increased TGF-β1 does.


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)

B2M levels decrease in neurogenic and myogenic niches following systemic or local attenuation of TGF-β signalingA. qRT-PCR quantification of B2m 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 < 0.04), and error bars indicate standard error of the mean (n = 4 old, 4 young). B. Brain sections spanning the hippocampus from young mice (2 month), as well as Alk5 inhibitor or vehicle treated old (24 month) mice, were immunostained for B2M (red), with Hoechst (blue) labeling all cell nuclei. Representative images are shown. Scale bar = 50 μM. C. Average B2M pixel intensity was quantified using MetaXpress software, and significant differences were identified by Student's t-tests (two-tailed) (*p < 3 × 10−6, **p < 0.0001). Error bars indicate standard error of the mean (n = 5 young, 3 old + Alk5i, 3 old + vehicle biological replicates (mice), with n = 12 technical replicates per mouse) D. Tibialis anterior muscle collected 5 days post-injury were immunostained for B2M (red), with Hoechst (blue) labeling all cell nuclei. Representative images are shown. Scale bar = 100 μM. E. Average B2M pixel intensity of 10 μM tibialis anterior muscle sections throughout sites of injury were quantified using MetaXpress software. Significant differences were identified by Student's t-tests (two-tailed) (*p < 7 × 10−7, **p < 5 × 10−5), and error bars indicate standard error of the mean (n = 3 biological replicates per group (mice), with n = 12 technical replicates per mouse) F. TA muscles collected 5 days post injury were immunostained for B2M (red), with Hoechst (blue) labeling all cell nuclei. Representative images are shown. Scale bar = 100 μM. G. Average B2M pixel intensity of TA muscle sections throughout sites of injury were quantified using MetaXpress software. Significant differences were identified by Student's t-tests (two-tailed) (*p < 0.002). Error bars indicate standard error of the mean (n = 3 biological replicates per group (mice), with n = 12 technical replicates per mouse).
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Figure 7: B2M levels decrease in neurogenic and myogenic niches following systemic or local attenuation of TGF-β signalingA. qRT-PCR quantification of B2m 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 < 0.04), and error bars indicate standard error of the mean (n = 4 old, 4 young). B. Brain sections spanning the hippocampus from young mice (2 month), as well as Alk5 inhibitor or vehicle treated old (24 month) mice, were immunostained for B2M (red), with Hoechst (blue) labeling all cell nuclei. Representative images are shown. Scale bar = 50 μM. C. Average B2M pixel intensity was quantified using MetaXpress software, and significant differences were identified by Student's t-tests (two-tailed) (*p < 3 × 10−6, **p < 0.0001). Error bars indicate standard error of the mean (n = 5 young, 3 old + Alk5i, 3 old + vehicle biological replicates (mice), with n = 12 technical replicates per mouse) D. Tibialis anterior muscle collected 5 days post-injury were immunostained for B2M (red), with Hoechst (blue) labeling all cell nuclei. Representative images are shown. Scale bar = 100 μM. E. Average B2M pixel intensity of 10 μM tibialis anterior muscle sections throughout sites of injury were quantified using MetaXpress software. Significant differences were identified by Student's t-tests (two-tailed) (*p < 7 × 10−7, **p < 5 × 10−5), and error bars indicate standard error of the mean (n = 3 biological replicates per group (mice), with n = 12 technical replicates per mouse) F. TA muscles collected 5 days post injury were immunostained for B2M (red), with Hoechst (blue) labeling all cell nuclei. Representative images are shown. Scale bar = 100 μM. G. Average B2M pixel intensity of TA muscle sections throughout sites of injury were quantified using MetaXpress software. Significant differences were identified by Student's t-tests (two-tailed) (*p < 0.002). Error bars indicate standard error of the mean (n = 3 biological replicates per group (mice), with n = 12 technical replicates per mouse).
Mentions: We compared the B2M expression levels as an indicator of inflammatory response in young and old mice treated with vehicle control, and old mice treated with the Alk5 inhibitor (Figure 4). B2M levels were extremely low in young brain and muscle, consistent with prior work [30], but increased significantly with aging (Figure 7A-7E). Importantly, when TGF-β signaling was attenuated in vivo by the Alk5 inhibitor, B2M levels were significantly diminished both in old brain (Figure 7B, 7C) and old muscle (Figure 7D, 7E). These results demonstrate that B2M becomes upregulated with aging in multiple tissues (suggesting an increase in inflammation) and that down-modulation of TGF-β signaling, which rejuvenates myogenesis and neurogenesis, normalizes B2M in myogenic and neurogenic regions to their young levels, suggesting attenuation of inflammation. As further support of this conclusion, the levels of B2M were also significantly reduced in regenerating regions of the old muscle administered with the dnTGFBR2, as compared to tissue administered with control GFP virus (Figure 7F, 7G). Furthermore, addition of low concentrations of TGF-β1 (0–5 ng/mL) to immune cells, specifically BV2 cells – a microglia cell line – did not affect B2M levels, as assayed by pixel intensity and percent area staining of B2M (Supplemental Figure 6A-6C). In contrast, high TGF-β1 (50 ng/mL) levels induced a significant increase in B2M expression. Thus, physiologically young levels TGF-β1 does not induce B2M, but increased TGF-β1 does.

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