GSK3β Inhibition Restores Impaired Neurogenesis in Preterm Neonates With Intraventricular Hemorrhage

Abstract

Intraventricular hemorrhage (IVH) is a common complication of prematurity in infants born at 23-28 weeks of gestation. Survivors exhibit impaired growth of the cerebral cortex and neurodevelopmental sequeale, but the underlying mechanism(s) are obscure. Previously, we have shown that neocortical neurogenesis continues until at least 28 gestational weeks. This renders the prematurely born infants vulnerable to impaired neurogenesis. Here, we hypothesized that neurogenesis is impaired by IVH, and that signaling through GSK3β, a critical intracellular kinase regulated by Wnt and other pathways, mediates this effect. These hypotheses were tested observationally in autopsy specimens from premature infants, and experimentally in a premature rabbit IVH model. Significantly, in premature infants with IVH, the number of neurogenic cortical progenitor cells was reduced compared with infants without IVH, indicating acutely decreased neurogenesis. This finding was corroborated in the rabbit IVH model, which further demonstrated reduction of upper layer cortical neurons after longer survival. Both the acute reduction of neurogenic progenitors, and the subsequent decrease of upper layer neurons, were rescued by treatment with AR-A014418, a specific inhibitor of GSK3β. Together, these results indicate that IVH impairs late stages of cortical neurogenesis, and suggest that treatment with GSK3β inhibitors may enhance neurodevelopment in premature infants with IVH.

Keywords: GSK3β; Pax6; Tbr2; intermediate progenitors; intraventricular hemorrhage; neurogenesis.

Figure 1.

Occurrence of IVH reduced all cycling (Ki67⁺) and total Tbr2⁺ cells in human preterm infants. (A) Representative immunofluorescence of cryosections from preterm infants with and without IVH of 23 weeks gestation (as indicated) labeled with Ki67 and Tbr2/Sox2 specific antibodies. Upper panel is low power image and lower panel is high magnification images of the boxed area in the upper panel. Note diminished number of all cycling and total Tbr2⁺ cells in infants with IVH relative to controls without IVH. (B–D) The bar charts are mean ± s.e.m. (n = 5 each). The total Tbr2⁺ cells were reduced in infants with IVH compared with controls without IVH. Sox2⁺ cells were comparable between infants with IVH and without IVH. Total Ki67⁺ cells were reduced in infants with IVH compared with controls without IVH. *P < 0.05 indicate comparison between infants with and without IVH.

Figure 2.

Reduction in the population of radial glia and IPCs as a function of postnatal age in E28.5 rabbits. (A) Representative immunofluorescence of cryosections from E28.5 rabbit kits labeled with Sox2 and Ki67 specific antibodies at D1 and D7 (as indicated). Note reduced expression of total and cycling Sox2+ cells in the VZ and SVZ of the dorsal telencephalon of rabbits at D7 compared with D1. The bar charts are mean ± s.e.m. (n = 5 each). The number of total and cycling Sox2⁺ radial glia were reduced as a function of postnatal age from D1 through D7. (B) The coronal cryosections from forebrain of E28.5 kits at D1 and 7 were labeled with Tbr2 and Ki67 specific antibodies. Upper panel is low power image and lower panel is high magnification images of the boxed area in the upper panel. Note abundance of Tbr2⁺ cells in the SVZ (as indicated) in E28.5 kits at D1 and scarcity of Tbr2 at D7. The bar charts are mean ± s.e.m. (n = 5 each). The number of total and cycling Tbr2⁺ IPC were reduced as a function of postnatal age from D1 through D7. (C) Representative Western blot analyses for Sox2 and Tbr2 on forebrain homogenates of preterm rabbits at D1, D3, and D7. The bar charts are mean ± s.e.m. (n = 5 each). Values were normalized to β actin levels. Both Sox2 and Tbr2 levels were reduced in kits at D3 and D7 relative to D1. **P < 0.01: indicate comparison between rabbits at D3 and D7 versus D1. Scale bar as indicated.

Figure 3.

Occurrence of IVH reduced Sox2 and Tbr2 in preterm rabbits. (A) Coronal brain slice from the frontoparietal lobe of E28.5 rabbit kits which show slit like ventricles in kits without IVH (upper panel) and moderate to severe IVH resulting in fusion of the lateral ventricles (middle and lower panel). Scale bar, 1 cm. (B–E) Representative immunofluorescence of cryosections from E28.5 rabbit kits with and without IVH at D3 (as indicated) labeled with ki67 and Tbr2/Sox2 specific antibodies. Upper panel is low power image and lower panel is high magnification images of the boxed area in the upper panel. Note diminished number of Tbr2⁺ and Sox2⁺ cells in rabbits with IVH relative to controls without IVH. The bar charts are mean ± s.e.m. (n = 5 each). The total and cycling Tbr2⁺ cells were reduced in rabbits with IVH compared with glycerol controls without IVH at D3, not at D7. All Sox2⁺ cells were reduced in rabbits with IVH compared with glycerol controls without IVH at both D3 and D7. Cycling Sox2⁺ cells were reduced in rabbits with IVH at D3, not at D7. (F) Representative Western blot analyses for Tbr2 and Sox2 on brain homogenates of preterm rabbits with and without IVH at D3 and D7. The bar charts are mean ± s.e.m. (n = 5 each). Values were normalized to β actin levels. Both Sox2 and Tbr2 levels were reduced in kits with IVH relative to controls without IVH at D3 and D7. ***P < 0.001, **P < 0.01, *P < 0.05 indicate comparison between rabbits with and without IVH at D3 and D7. Scale bar as indicated.

Figure 4.

IVH reduced number of neurons in the upper cortical layer. (A) Representative immunofluorescence of cryosections from preterm rabbits with and without IVH at D14 (as indicated) labeled with Cux1 and Satb2 specific antibodies. Upper panel is Cux1 and lower panel is merge image of Cux1 and Satb2 form upper cortical layer as indicated. Note reduced number of Cux1+ and Satb2+ neurons in rabbits with IVH compared with controls without IVH. The bar charts are mean ± s.e.m. (n = 5 each). Stereological quantification revealed that both Cux1⁺ and Satb2⁺ neurons were reduced in rabbits with IVH compared with controls. (B) Representative Western blot analyses for Satb2 on brain homogenates of preterm rabbits with and without IVH at D14. The bar charts are mean ± s.e.m. (n = 5 each). Values were normalized to β actin levels. Satb2 levels were reduced in rabbits with IVH relative to controls. **P < 0.01, ***P < 0.001 indicate comparison between infants with and without IVH.

Figure 5.

IVH increases Pax6 levels and reduces phosphorylation of retinoblastoma protein. (A) The mRNA expressions of Tbr2, Pax6, NeuroD1, NeuroD6, Satb2, CYCLIN D2, and CDK6 were quantified by real time-qPCR using TaqMan probes in rabbits with and without IVH at D3 and D7. Note reduced expression of Tbr2 at D3 and elevated levels of Pax6 at D3 in rabbits with IVH relative to controls without IVH. The expression of NeuroD1, NeuroD6, and Satb2 were comparable between rabbits with and with IVH at both D3 and D7. (B) Representative Western blot analyses for Pax6, cyclin D1, cyclin D2, CDK6, Ngn2, p-Rb (S807/811), and p-Rb (S780) on brain homogenates of preterm rabbits with and without IVH at D3 and 7. The bar charts are mean ± s.e.m. (n = 5 each). Values were normalized to β actin levels. Pax6 levels were increased in rabbits with IVH relative to controls at D7; and p-Rb (S807/811) levels were reduced in rabbits with IVH at D7. *P < 0.05, **P < 0.01 indicate comparison between infants with and without IVH for D3 or D7.

Figure 6.

AR-A014418 (ARA) treatment enhances neurogenesis. (A, B) Representative immunofluorescence of cryosections from preterm rabbits with vehicle and ARA treatment at D7 (as indicated) labeled with Ki67 and Tbr2/Sox2 specific antibodies. Upper panel is low power image and lower panel is high magnification images of the boxed area in the upper panel. Note abundance of Tbr2⁺ and Sox2+ cells in ARA-treated kits with IVH compared with vehicle controls with IVH. The bar charts are mean ± s.e.m. (n = 5 each).The total number of Tbr2⁺ IPCs were increased in ARA-treated kits compared with Vehicle controls at D7. The total and cycling Sox2⁺ cell were higher in ARA-treated kits compared with vehicle controls at D3. (C) Western blot analyses was performed on brain homogenates of preterm rabbits with and without IVH using Tbr2 and Sox2 specific antibodies. Sox2 expression was increased in ARA-treated rabbits compared with vehicle controls with IVH at D3. Tbr2 levels were comparable between groups. **P < 0.01, ***P < 0.01 indicate comparison between ARA and vehicle treated kits with IVH for D3 or D7.

Figure 7.

ARA treatment increases the number of neurons in the upper cortical layer. (A) Representative immunofluorescence of cryosections from preterm rabbits with IVH treated with ARA or vehicle at D14 (as indicated) labeled with Cux1 and Satb2 specific antibodies. Upper panel is Cux1 and lower panel is merge image of Cux1 and Satb2 form upper cortical layer as indicated. Note increased number of Cux1⁺ and Satb2⁺ neurons in ARA-treated rabbits with IVH compared with vehicle controls. The bar charts are mean ± s.e.m. (n = 5 each). Stereological quantification revealed that both Cux1⁺ and Satb2⁺ neurons were increased in ARA-treated rabbits with IVH compared with vehicle controls. (B) Representative Western blot analyses for Cux1 and Satb2 on brain homogenates of preterm rabbits with IVH treated with ARA or vehicle at D14. The bar charts are mean ± s.e.m. (n = 5 each). Values were normalized to β actin levels. Satb2 and Cux1 levels were increased in ARA-treated rabbits with IVH relative to vehicle controls. *P < 0.05, **P < 0.01, ***P < 0.001 indicate comparison between ARA and vehicle treated kits with IVH for D3 or D7.

Figure 8.

GSK3-β inhibition increases Tbr2 and NeuroD1 mRNA, and reduces Pax6 protein expression. (A) The mRNA expressions of Tbr2, Pax6, NeuroD1, NeuroD6, Satb2, Sox5, cyclin D2, and CDK6 were quantified by Real time-qPCR using TaqMan probes in ARA and vehicle treated rabbits with IVH at D3 and D7. Note increased expression of Tbr2 at D3 and NeuroD1 at D7 in ARA-treated rabbits with IVH relative to vehicle controls with IVH. The expression of Pax6, NeuroD1, NeuroD6, Satb2, Sox5, cyclin D2, and CDK6 were comparable between ARA and vehicle treated rabbits with IVH at both D3 and D7. (B) Representative Western blot analyses for Pax6, cyclin D1, cyclin D2, CDK6, pRB (S807/811), and pRB (S780) on brain homogenates of preterm rabbits with and without IVH at D3 and 7. The bar charts are mean ± s.e.m. (n = 5 each). Values were normalized to β actin levels. Pax6 levels were reduced in ARA-treated rabbits with IVH relative to vehicle controls at D3. *P < 0.05, **P < 0.01 indicate comparison between ARA and vehicle treated kits with IVH for D3 or D7.