Abstract
Purpose:
CLOCK and NPAS2, homologous circadian clock proteins, are expressed in the mammalian retina. However, their specific roles in retinal gene regulation or function have not been elucidated. This study was conducted to determine whether NPAS2 and CLOCK are co-expressed in retinal neurons and their effects on retinal gene expression and function.
Methods:
Studies were performed using C57BL/6 wildtype (WT), Clock-/-, and Npas2-/- mice. Laser capture microdissection and quantitative real-time PCR were performed to isolate the ganglion cell layers (GCL) at five time points for transcription expression analyses for Npas2, Clock, and Adcy1. Luciferase reporter assay in NG108-15 cells was conducted to determine whether CLOCK/BMAL1 and/or NPAS2/BMAL1 heterodimers could activate the Adcy1 promoter. Contrast sensitivity was measured using optokinetic tracking at mid-day and mid-night time points, and scotopic and photopic electroretinograms (ERG) were recorded to measure retinal responses to light.
Results:
CLOCK and NPAS2 co-localized in a subset of retinal ganglion cells. Npas2 transcripts were rhythmic in the GCL of WT mice, but were damped or arrhythmic in Clock-/- mice. Clock transcripts in the GCL of WT and Npas2-/- mice were similarly expressed. In both Npas2-/- and Clock-/- mice, the Adcy1 transcript rhythm was abolished in the GCL. When co-expressed with BMAL1 in NG108-15 cells, NPAS2 was more efficacious at activating the Adcy1 promoter. While diminished contrast sensitivity rhythm was observed in Npas2-/- mice, the contrast sensitivity rhythm was abolished in Clock-/- mice. Significant deficiencies in both scotopic and photopic ERG amplitudes were observed in Clock-/- mice, but not in Npas2-/- mice.
Conclusions:
These results indicate that NPAS2 and CLOCK are co-expressed in a subset of retinal ganglion cells. CLOCK appears to regulate the rhythmic expression of Npas2 transcripts, but NPAS2 deficiency does not appear to affect the expression of Clock transcripts in GCL. NPAS2 is necessary for the rhythmic expression of Adcy1 transcripts in the GCL, and our luciferase reporter assay results suggest that there is a cell-specific mechanism that confers specificity to the NPAS2/BMAL1 heterodimer. CLOCK appears to control contrast sensitivity rhythm in part through regulating NPAS2 expression in the GCL. NPAS2, unlike CLOCK, does not appear to affect retinal function outside the GCL.
Keywords: 458 circadian rhythms •
531 ganglion cells •
478 contrast sensitivity