Abstract
Purpose: :
Crystallins are structural proteins of the lens of the eye. Once believed to be lens-specific, many, if not all, crystallins are present in other tissues and it is now believed that crystallins evolved for specialized function in the lens from pre-existing proteins with other functions. Our studies with the Nuc1 rat demonstrated that βA3/A1-crystallin is expressed only in astrocytes in the neural retina. Nuc1 results from a mutation in the βA3/A1-crystallin gene. The astrocyte changes that we report here reflect the loss of the "original" or "non-crystallin" function of βA3/A1-crystallin.
Methods: :
Astrocytes were cultured from 2 day old brain of Nuc1 and wildtype (WT) rats. Expression of glial fibrillary acidic protein (GFAP) in retinal flatmounts and cultured astrocytes of Nuc1 and WT rats was assessed by confocal microscopy and western blotting. Ultrastructure of Nuc1 astrocytes was assessed by transmission electron microscopy (TEM). Microarray studies utilized the Affymatrix system.
Results: :
Confocal microscopy of retinal flatmounts labeled with GFAP, clearly showed changes in process length in Nuc1 astrocytes. The normal stellate appearance of astrocytes with delicate processes was not observed in the Nuc1 retina. In addition, TEM of retinal astrocytes from WT and Nuc1 rats shows that in the mutants the IF arrays have abnormal size and organization. The cultured astrocytes from Nuc1 also show that the nucleus is irregular in shape and there are clear breaks in the nuclear envelope. Ribosomes are much more dispersed and less compact in the Nuc1 astrocytes. Our microarray studies show that GFAP mRNA actually increases in Nuc1, however, the level of GFAP protein in Nuc1 astrocytes is greatly reduced.
Conclusions: :
Our data suggest, for the first time, that βA3/A1-crystallin is essential for the normal functioning of retinal astrocytes. In addition, the involvement of βA3/A1-crystallin in normal astrocyte function and subsequent vascular development of the eye suggests a previously unknown non-lens function for this protein. Our data implicate βA3/A1-crystallin as an important regulatory factor that mediates its effect through disruption of normal regulation of IFs. IFs are now known to be critical elements in a variety of regulatory processes by serving as scaffolds to sequester or organize molecules of signaling pathways. One major regulatory function in which IFs have been implicated is the control of cell death via apoptosis and impaired apoptosis may be a key factor in the Nuc1 phenotype.
Keywords: astrocytes: optic nerve head • development • crystallins