Growth Differentiation Factor 11 treatment leads to neuronal and vascular improvements in the hippocampus of aged mice

Abstract

Aging is the biggest risk factor for several neurodegenerative diseases. Parabiosis experiments have established that old mouse brains are improved by exposure to young mouse blood. Previously, our lab showed that delivery of Growth Differentiation Factor 11 (GDF11) to the bloodstream increases the number of neural stem cells and positively affects vasculature in the subventricular zone of old mice. Our new study demonstrates that GDF11 enhances hippocampal neurogenesis, improves vasculature and increases markers of neuronal activity and plasticity in the hippocampus and cortex of old mice. Our experiments also demonstrate that systemically delivered GDF11, rather than crossing the blood brain barrier, exerts at least some of its effects by acting on brain endothelial cells. Thus, by targeting the cerebral vasculature, GDF11 has a very different mechanism from that of previously studied circulating factors acting to improve central nervous system (CNS) function without entering the CNS.

Figure 1

Systemic GDF11 treatment enhances neurogenesis in the hippocampus of old mice. (a) Representative confocal images showing the effects of systemic GDF11 treatment on BrdU⁺/NeuN⁺ newborn neurons in the GCL of old mice. White arrows indicate representative cells that are positive for both markers. (b) Quantification of BrdU⁺/NeuN⁺ newborn neurons in GCL (total area). n = 4 for each experimental group. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, **p = 0.005 compared to vehicle control. (c) Representative confocal images showing the effects of systemic GDF11 treatment on Sox2⁺ Type1 neural stem cells in the GCL of old mice. White arrows indicate representative cells that are positive for the marker. (d) Quantification of Sox2⁺ Type1 neural stem cells in GCL (total area). n = 8 for each experimental group. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, ***p = 0.001 compared to vehicle control. (e) Representative confocal images showing the effects of systemic GDF11 treatment on DCX⁺ neural progenitor/immature neurons in the GCL of old mice. White arrows indicate representative cells that are positive for the marker. (f) Quantification of DCX⁺ neural progenitor/immature neurons in GCL (total area). n = 8 for each experimental group. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, *p = 0.03 compared to vehicle control.

Figure 2

Systemic GDF11 treatment improves vasculature in the hippocampus and cortex of old mice. (a) Representative confocal images showing the effects of systemic GDF11 treatment on blood vessels in the dentate gyrus of old mice. (b) Measurement of blood vessel-occupied area (per field of view). n = 7 for each experimental group. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, *p = 0.05 compared to vehicle control. (c) Measurement of number of blood vessels (per field of view). n = 7 for each experimental group. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, *p = 0.02 compared to vehicle control. (d) Measurement of number of blood vessel endpoints (per field of view). n = 7 for each experimental group. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, *p = 0.03 compared to vehicle control. (e) Representative confocal images showing the effects of systemic GDF11 treatment on blood vessels in the frontal cortex of old mice. (f) Measurement of blood vessel-occupied area (per field of view). n = 8 for each experimental group. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, *p = 0.04 compared to vehicle control.

Figure 3

Systemic GDF11 treatment increases neuronal activity markers in the hippocampus and cortex of old mice. (a) Representative confocal images showing the effects of systemic GDF11 treatment on DeltaFosB levels in the GCL of old mice. White arrows indicate representative cells that are positive for the marker. Corresponding thresholded images with counted cells highlighted in blue are shown on the right. (b) Quantification of DeltaFosB⁺ cells and measurement of DeltaFosB mean signal intensity per cell in GCL (total area). n = 8 for each experimental group. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, *p = 0.04 compared to vehicle control. (c) Representative confocal images showing the effects of systemic GDF11 treatment on VGLUT1 levels in the frontal cortex of old mice. (d) Measurement of % VGLUT1⁺ area and VGLUT1 mean signal intensity (per field of view). n = 4 for each experimental group. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, *p = 0.04 compared to vehicle control.

Figure 4

Multiple types of analyses show that GDF11 does not cross the BBB. (a) GDF11 levels in the serum of 24-month-old mice following acute GDF11 treatment. Full-length blot is presented in Supplementary Fig. 7a. (b) ELISA of SMAD2/3 phosphorylation in whole tissue lysates of 24-month-old mice following acute GDF11 treatment (1 mg/kg). n = 6 for each experimental group. Data plotted as the background-subtracted absorbance and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, *p = 0.03 (heart), ***p = 0.0003 (kidney), ***p = 0.0006 (liver), **p = 0.01 (spleen), ^#p = 0.06 (muscle), not significant (ns) (brain) compared to vehicle controls of each tissue type. (c) ELISA of SMAD2/3 phosphorylation in primary mouse cortical neurons and primary mouse astrocytes following 1-hour treatment with GDF11 (50 ng/ml) or vehicle. n = 6 for each neuron condition, n = 4 for each astrocyte condition. Data calculated as fold change from vehicle controls and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, ***p = 0.0001 (neuron), ^#p = 0.06 (astrocyte), compared to vehicle controls of each cell type. (d) Number of pSMAD2/3⁺ nuclei in SVZ-derived, dissociated neurospheres following 90-minute treatment with GDF11 (50 ng/ml) or vehicle. n = 3 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, *p = 0.01 compared to vehicle control. (e) Average colony size (number of cells per sphere) of young and old SVZ-derived neurospheres following 10-day treatment with GDF11 (50 ng/ml) or vehicle, in a clonal assay. n = 11 for young, n = 3 for old condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, *p = 0.02 compared to vehicle control. (f) Number of Tuj1+ cells differentiated from young and old SVZ-derived neurospheres following 7-day treatment with GDF11 (50 ng/ml) or vehicle, in a differentiation assay. n = 4 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, *p = 0.04, **p = 0.006 compared to vehicle control. (g) Detection of biotinylated recombinant GDF11 with streptavidin-HRP (left) or Coomassie staining (right). Full-length blot and gel are presented in Supplementary Fig. 7b. (h) Biotinylated GDF11 levels in the brain parenchyma (left) and the spleen (right) of 3–4-month-old mice following acute GDF11 treatment (8 mg/kg). Biotinylated recombinant GDF11 protein was loaded to help detect the biotinylated protein in tissue samples. Tubulin was used as a loading control. Full-length blots are presented in Supplementary Fig. 7c.

Figure 5

Endothelial cells of the cerebral vasculature are responsive to GDF11. (a) ELISA of SMAD2/3 phosphorylation in mBVECs following 1-hour treatment with GDF11 (50 ng/ml) or vehicle. n = 4 for each condition. Data calculated as fold change from vehicle control and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, *p = 0.03 compared to vehicle control. (b) ELISA of serum VEGF levels following systemic GDF11 or vehicle treatment in old mice. n = 8 for each experimental group. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, *p = 0.03 compared to vehicle control. (c) ELISA of VEGF levels in mBVEC conditioned medium following 24- or 72-hour treatment with GDF11 (50 ng/ml), GDF8 (50 ng/ml), TGFβ2 (50 ng/ml) or vehicle. n = 3 for each condition. Data calculated as fold change from vehicle controls and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test *p = 0.02, **p = 0.002 compared to vehicle control at designated time point. (d) qPCR analysis of Vegf and Kdr gene expression following 72-hour treatment of mBVECs with GDF11 (50 ng/ml) or vehicle. n = 3 for each condition. Data calculated as fold change from vehicle controls and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, **p = 0.001 (Vegf) and *p = 0.05 (Kdr) compared to vehicle controls of each gene. (e) ELISA of SMAD2/3 phosphorylation in hBVECs following 1-hour treatment with GDF11 (50 ng/ml) or vehicle. n = 4 for each condition. Data calculated as fold change from vehicle control and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, ****p = 0.0001 compared to vehicle control. (f) ELISA of VEGF levels in hBVEC conditioned medium following 48-hour treatment with GDF11 (50 ng/ml) or vehicle. n = 6 for each condition. Data calculated as fold change from vehicle control and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t-test, *p = 0.02 compared to vehicle control.