Circadian Clock Components and Outputs in a mammalian retinal ganglion cell line

NIETO PAULA S; VALDEZ DIEGO J; GUIDO MARIO E

Fort Lauderdale-Miami- EE.UU.

Congreso; The Association for Research in Vision and Ophthalmology (ARVO); 2007

INTRODUCTION Retinal oscillators may act like clocks to induce changes in the visual system according to the phase of the day by predicting environmental changes. The oscillatory and photoreceptive capacities are likely to converge all together on selected retinal cell populations such as the retinal ganglion cells (RGCs) in the mammalian retina as previously shown by us in the avian RGCs (Garbarino et al., 2004a-b, Contin et al., 2006). Recently, Dr. Agarwal’s group establishes a clonal retinal cell line from virally transformed dispersed postnatal rat retinal cells, named RGC-5 which has certain characteristics of retinal ganglion cells (RGCs) based on expression of specific markers such as Thy-1, Brn-3c; Neuritin and NMDA receptor, and on sensitivity to glutamate excitotoxicity and neurotrophin withdrawal (Krishnamoorthy et al., 2001). PURPOSE: To investigate the potential capacity of mammalian retinal ganglion cells (RGCs) as autonomous circadian oscillators, we utilized a transformed mammalian retinal cell line (RGC-5) which has certain characteristics of RGCs based on expression of specific markers (thy-1, brn-3c, neuritis and NMDA receptor) and glutamate sensitivity (Krishnamoorthy et al., 2001). To do this, we assessed in these cultured cells, expression of clock proteins (Per, Cry and Clock), photopigments (rhodopsin and melanopsin) and a clock output, the key melatonin synthesizing enzyme, serotonin N-acetyl transferase (NAT). METHODS. RGC-5 were grown in basal culture medium DMEM and 10% BFS to about 80% of confluence, arrested during 24 h and synchronized by a 2 h-serum shock with 50% BFS at time 0 (zeitgeber time, ZT 0) according to Balsalobre´s protocol (1998). Then, the medium was replaced by 10% BSF and cells were collected at different ZTs during the following 24 h. Cells were resuspended in 1% PBS buffer for protein immunocharacterization with specific clock protein antibodies by Western blot, or homogenized in TRIZol (Gibco) for RNA extraction and RT-PCR analysis.We previously investigated the response of the RGC-5 cells to different effectors such as staurosporine (SS). We found that SS, a protein kinase inhibitor; induce cell differentiation at a lower range of concentration than those previously reported by Frassetto et al. (2006). Concentrations ranging from 10 to 100 ng/mL are sufficient to induce cell arrest and neurite outgrowth; however, 24 hours post SS administration, cells dye by apoptosis in a dose dependent manner according to the nuclear fluorescent staining with DAPI. Because of this, all consecutive experiments were performed with proliferating cells (Fig. 1). We found that cells of the RGC-5 line present detectable levels of expression for Per1, Per2 and Cry proteins with a temporal variation (p<0.039 by ANOVA) exhibiting higher levels at ZTs 8-12 after synchronization by serum shock and coger values at ZTs 16-20 (Fig. 2).  They did express the mRNAs for Clock (Fig. 2) and AA-NAT (Fig. 3). In addition, very low levels of AA-NAT enzyme activity were observed in the cultures as compared with levels found in the pineal gland and whole retina. These cells did not show detectable levels of rhodopsin or melanopsin mRNAs (Fig. 3).CONCLUSIONS. The results show that cultures of RGC-5 cells contain the oscillatory circadian machinery expressing different clock proteins (PER1, PER2, CRY1 and Clock) together with a clock output, the melatonin synthesizing enzyme AA-NAT. Based on these observations, we may infer that the physiology of these mammalian retinal cells may also be controlled by an endogenous circadian clock.