Glaucoma Inducing Retinal Ganglion Cell Degeneration Alters Diurnal Rhythm of Key Molecular Components of the Central Clock and Locomotor Activity in Mice

Glaucoma is a chronic optic neuropathy characterized by the progressive de- generation of retinal ganglion cells (RGC). These cells play a crucial role in transmitting visual and non-visual information to brain regions, including the su- prachiasmatic nucleus (SCN), responsible for synchronizing biological rhythms. To understand how glaucoma affects circadian rhythm synchronization, we in- vestigated potential changes in the molecular clock machinery in the SCN. We found that the progressive increase in intraocular pressure (IOP) negatively correlated with spontaneous locomotor activity (SLA). Transcriptome analy- sis revealed significant alterations in the SCN of glaucomatous mice, including downregulation of genes associated with circadian rhythms. In fact, we showed a loss of diurnal oscillation in the expression of vasoactive intestinal peptide (Vip), its receptor (Vipr2), and period 1 (Per1) in the SCN of glaucomatous mice. These findings were supported by the 7-h phase shift in the peak expression of arginine vasopressin (Avp) in the SCN of mice with glaucoma. Despite maintaining a 24-h period under both light/dark (LD) and constant dark (DD) conditions, glaucoma- tous mice exhibited altered SLA rhythms, characterized by decreased amplitude. Taken altogether, our findings provide evidence of how glaucoma affects the regulation of the central circadian clock and its consequence on the regulation of circadian rhythms. Overall design: We used the DBA2J strain as a model of glaucoma development. These mice carry a spontaneous mutation in the GPNMB (transmembrane glycoprotein) gene resulting in high IOP and retinal damage (Anderson et al., 2001). As control mice we used DBA/2J-Gpnmb+SjJ that do not show GPNMB mutation (Jackson Laboratory (Stock No: 007048 and 000671). Experimental procedures were conducted in accordance with national and international ethical guidelines and regulations and approved by the Ethical Committee for Animal Use (ICB/USP, number 8143290819). Mice were group housed (2 to 5 mice per cage) at 25°C ± 1 in a 12h light: 12h dark (12:12 LD) cycle. The light source consisted of LED lights providing illumination at 400 lux (wavelength range from 420 to 750 nm). Food and water were available ad libitum throughout the study. The lights were turned on at 7 AM (zeitgeber time - ZT0) and turned off at 7 PM (ZT12). Control and advanced glaucoma (n=3-4) mice were euthanized for SCN microdissection, which was used for RNA isolation and transcriptome analysis. Sections of 600 µm were obtained in a cryostat from the hypothalamic region, where the SCN is located (Bregma -0.22 mm to -0.82 mm), according to the coordinates of Paxinos & Watson atlas (The Mouse Brain in Stereotaxic Coordinates, 2008).