Active Notch Is Decreased in Astrocytes With Mutation in βA3/A1-Crystallin

Abstract

Purpose: : Astrocytes in the Nuc1 rat and in the human disease, PFV, surround the persistent hyaloid vasculature [Dev. Dyn., 2005]; in the normal eye, astrocytes are not associated with the hyaloid. The Nuc1 gene, βA3/A1-crystallin, is expressed in astrocytes [Mol. Cell Neurosci., 2008] and Notch is the most significantly enriched signaling pathway in developing astrocytes. We have investigated a possible role of βA3/A1-crystallin in Notch signaling in developing astrocytes.

Methods: : Expression of Notch proteins in the astrocytes of Nuc1 and wildtype (WT) rats was assessed by western blotting. Myc epitope-tagged Notch1-IC and mutants were used to verify translocation of active Notch. Microarray analysis was performed using the Affymetrix system; Ingenuity pathway analysis software identified functional groups of genes differentially regulated in Nuc1 astrocytes. TIRF microscopy visualized the actin cytoskeleton in migrating wildtype and Nuc1 astrocytes transfected with pEGFP-actin.

Results: : Active Notch 1 is decreased in Nuc1 astrocyte cell extracts relative to WT. Translocation of active Notch to the nucleus is inhibited in Nuc1 astrocytes. Based on microarray analysis, different functional groups of genes were combined into networks. Major network changes observed included astrocyte and blood vessel development and cell-cell interactions. TIRF studies indicate that Nuc1 astrocytes move more slowly and randomly than WT astrocytes.

Conclusions: : These data suggest, for the first time, that abnormal expression of βA3/A1-crystallin affects Notch signaling and migration of astrocytes in the developing retina. Abnormal expression of Notch in the retina results in an excessively dense network of poorly-organized, dysfunctional vessels; the Nuc1 retina shows a similar vascular pattern [Neuroscience, 2006 and Mol. Cell Neurosci., 2008]. Thus, βA3/A1-crystallin may have a role in the cross-talk between Notch and VEGF signaling pathways at the developing glia-vascular interface, potentially defining a "non-crystallin" function of the protein.

Keywords: crystallins • astrocyte • signal transduction