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Dysregulation of Astrocyte Extracellular Signaling in Costello Syndrome


Dysregulation of Astrocyte Extracellular Signaling in Costello Syndrome

Robert Krencik et al. Sci Transl Med.


Astrocytes produce an assortment of signals that promote neuronal maturation according to a precise developmental timeline. Is this orchestrated timing and signaling altered in human neurodevelopmental disorders? To address this question, the astroglial lineage was investigated in two model systems of a developmental disorder with intellectual disability caused by mutant Harvey rat sarcoma viral oncogene homolog (HRAS) termed Costello syndrome: mutant HRAS human induced pluripotent stem cells (iPSCs) and transgenic mice. Human iPSCs derived from patients with Costello syndrome differentiated to astroglia more rapidly in vitro than those derived from wild-type cell lines with normal HRAS, exhibited hyperplasia, and also generated an abundance of extracellular matrix remodeling factors and proteoglycans. Acute treatment with a farnesyl transferase inhibitor and knockdown of the transcription factor SNAI2 reduced expression of several proteoglycans in Costello syndrome iPSC-derived astrocytes. Similarly, mice in which mutant HRAS was expressed selectively in astrocytes exhibited experience-independent increased accumulation of perineuronal net proteoglycans in cortex, as well as increased parvalbumin expression in interneurons, when compared to wild-type mice. Our data indicate that astrocytes expressing mutant HRAS dysregulate cortical maturation during development as shown by abnormal extracellular matrix remodeling and implicate excessive astrocyte-to-neuron signaling as a possible drug target for treating mental impairment and enhancing neuroplasticity.

Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.


Fig. 1
Fig. 1. Precocious Ras-dependent astroglial maturation of CS-iPSCs
(A to C) Neuroepithelial development in three wild-type (WT) and four G12S iPSC lines. (D and E) Time course of astroglial development was measured by comparing the percent of cells in HRASWT lines (n = 3) and HRASG12S lines (n = 4) expressing astroglial progenitor–associated markers S100 (week 8, P = 0.007; week 16, P = 0.0023) and CD44 (week 8, P = 0.0462), as well as the astroglial marker GFAP (week 16, P = 0.025; week 24, P = 0.0274). (F) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) measurements of relative expression (n = 3 for each genotype) of astrocyte markers NFIA (week 16, P = 0.0279; week 24, P = 0.0404), S100B (week 16, P = 0.0209), CD44 (week 16, P = 0.0065), GFAP (week 16, P = 0.0413; week 24, P = 0.0161), and THBS1 (week 16, P = 0.0484; week 24, P = 0.0014). (G) Confocal imaging of THBS1- and AQP4-labeled astrocytes in all lines by 28 weeks. White box, magnified view. Scale bar, 50 mm. Data are displayed as means ± SEM in (C) and (E), and means ± SD in (F). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4′,6-diamidino-2-phenylindole.
Fig. 2
Fig. 2. Phenotypic changes induced by mutant HRAS in astrocytes
(A) pERK-labeled cells in HRASWT (n = 3) and HRASG12S (n = 3) cells (24-hour withdrawal, P = 0.006). (B) Apoptosis was determined in HRASWT lines (n = 3) and HRASG12S lines (n = 3) by assessing cleaved caspase 3 labeling (Cl. casp3). (C) Oxidative stress was determined by ROS-CellROX intensity in HRASWT lines (n = 3) and HRASG12S lines (n = 3, P = 0.0318). (D) BrdU labeling measurements for the indicated times after uptake as a marker of proliferation (in the presence of EGF and FGF2, P = 0.003; with CNTF for 3 days, P = 0.0164). (E and F) Ki67-labeled cells (E) and β-galactosidase (β-gal) activity–containing cells (F) (P = 0.009) after 8 days in the presence of CNTF. (G) Relative cell area measured by viral-mediated GFP expression in the presence of EGF and FGF2 (n = 3 for each group, 30 cells each; P = 0.0006). (H) Yellow arrow, engraftment site of a human astrosphere into a hippocampal slice culture. HRASG12S-induced morphological complexity was maintained in hippocampal slice cultures. (I) Size comparison of Aldh1L1-GFP–positive astrocytes in p21 HRASWT and HRASG12V mouse somatosensory cortex (n = 45 cells from three mice for each; P = 0.0001). Scale bar, 50 μm. Data are displayed as means ± SD. hGFAP, human GFAP.
Fig. 3
Fig. 3. Altered transcript expression caused by overactivated Ras signaling is reversed by knockdown of SNAI2
(A) Heat maps of select microarray signals at 8 weeks show consistent increases in multiple functional categories (P ≤ 0.05, one-tailed t test) in the order from highest up-regulated on average. (B) Gene expression was compared in HRASG12S-expressing cells in the presence or absence of FTI-277 from 18 to 20 weeks of development (n = 3 for each group) (SNAI2, P = 0.0245; POSTN, P = 0.0002; MGP, P = 0.004; LUM, P = 0.0041; GPC6, P = 0.0167). (C) Extracellular periostin was also measured (P = 0.0005). (D and E) Effect of 2 weeks of SNAI2 knockdown compared against a scrambled siRNA sequence was measured in week 20 HRASG12S-expressing cells (n = 3 for each group) (D) by expression of the same set of genes (SNAI2, P = 0.0063; POSTN, P = 0.0001; MGP, P = 0.01; LUM, P = 0.0002; GPC6, P = 0.0175) and (E) by measuring the concentration of extracellular periostin (P = 0.0037). Data are displayed as means ± SD.
Fig. 4
Fig. 4. Enhanced extracellular signaling by HRAS mutant astroglial cells
(A) Effect of coculture of RGCs with either HRASWT or HRASG12S astrocytes (n = 3 for each genotype) on synapse area and density, as measured by the relative value of apposed of pre- and postsynaptic markers Syn1 and PSD-95 (P = 0.023). Yellow arrows, RGCs; white arrows, synapses; inset, magnified example. (B) Neurite length was measured 24 hours after RGCs were exposed to ACM and astrocyte-containing inserts from HRASWT or HRASG12S astrocytes (n = 3 for each, P = 0.023). Asterisks, cell bodies; arrows, growth cones. (C) Listing of proteins found to be overabundant in HRASG12S ACM as determined by AFE over HRASWT (n = 3 for each). (D) Glycoprotein-containing extracellular matrix on the Surface of HRASWT and HRASG12S astrocytes (expressed as WFA + pixels/number of cells) (n = 3 for each group) (P = 0.0289). (E) Accumulation of WFA-labeled cortical PNNs in mice with astrocytic expression of HRASWT or HRASG12V (n = 3 for each) (P = 0.0016) and the density of PV-labeled cells (P = 0.0395). (F) Abundance of protein in p21 mouse cortex (n = 3 of each genotype) as measured by Western blotting (GFAP, P = 0.0202; NCAN, P = 0.0306). (G) WFA-labeled PNN intensity in visual cortex (Vis) and somatosensory cortex (SS) after DR of HRASWT or HRASG12V mice (n = 3 of each, littermates) from p8 to p19 (HRASWT Vis compared to HRASG12S Vis, P = 0.0128; HRASWT SS compared to HRASG12S Vis, P = 0.4524). Scale bar, 50 μm. Significance was tested using unpaired t tests. Data are displayed as means ± SD.
Fig. 5
Fig. 5. Proposed model for dysfunction of RASopathic astrocytes
Overactivation of the Ras/MAPK signaling pathway in mutant HRAS (mtHRAS) astrocytes (green) results in both early Ras-specific gliogenesis and an extracellular response, which can be in part reduced by FTI-277 and SNAI2 knockdown. Subsequently, extracellular matrix remodeling occurs and leads to an experience-independent accumulation of PNNs surrounding inhibitory cortical neurons (gray), which may possibly alter critical period plasticity.

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