Ординатура / Офтальмология / Английские материалы / Recent Advances in Retinal Degeneration_LaVail, Hollyfield, Anderson _2008

Fig. 2 Phagocytosis by ARPE19 cell line. Confluent ARPE19 cells were challenged with ROSs for 4h followed by a chase in growth medium for 3h or over night. Total ROSs per field of view were counted

of myosin VIIa is thought to delay degradation of phagosomes by inhibiting their transport to basal RPE, where digestion occurs.

ARPE19 cells ingest (Finnemann et al., 1997) and digest ROSs more slowly than primary mouse RPE cells do. After a 4h pulse with ROSs, they show reduction in total opsin labeling after 3h and over night incubation in GM (Fig. 2).

4 Conclusions

This assay can be a valuable tool to analyze ingestion and digestion of ROS disk membranes by the RPE. By using primary RPE cultures, one can take advantage of mutant mouse models to study RPE-dependent retinal degeneration. We are using this system to dissect out roles for motor proteins in the transport of phagosomes and lysosomes in the RPE (e.g. Gibbs et al., 2003). We are also using it to help understand mechanisms underlying RPE pathologies; for example, in mouse models for macular dystrophy, such as Stargardt’s macular degeneration type 3 (Karan et al., 2005).

Acknowledgments This research was supported by NIH grant EY07042.

References

Bok, D., and Hall, M. O., 1971, The role of the pigment epithelium in the etiology of inherited retinal dystrophy in the rat, J. Cell Biol. 49:664–682.

Bosch, E., Horwitz, J., and Bok, D., 1993, Phagocytosis of outer segments by retinal pigment epithelium: phagosomelysosome interaction, J. Histochem. Cytochem. 41:253–263.

Chaitin, M. H., and Hall, M. O., 1983, Defective ingestion of rod outer segments by cultured dystrophic rat pigment epithelial cells, Invest. Ophthalmol. Vis. Sci. 24(7):812–20.

Colley, N. J., and Hall, M. O., 1986, Phagocytosis of lightand dark-adapted rod outer segments by cultured RPE cells: a reassessment, Exp. Eye Res. 42:323–329.

D’Cruz, P. M., Yasumura, D., Weir, J., Matthes, M. T., Abderrahim, H., LaVail, M. M., and Vollrath, D., 2000, Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat, Hum. Mol. Genet. 9(4):645–51.

Dunn, K. C., Aotaki-Keen, A. E., Putkey, F. R., and Hjelmeland, L. M., 1996, ARPE-19, a human retinal pigment epithelial cell line with differentiated properties, Exp. Eye Res. 62:155–169.

Feng, W., Yasumura, D., Matthes, M. T., LaVail, M. M., and Vollrath, D., 2002, Mertk triggers uptake of photoreceptor outer segments during phagocytosis by cultured retinal pigment epithelial cells, J. Biol. Chem. 277(19):17016–22.

Finnemann, S. C., Leung, L. W., and Rodriguez-Boulan, E., 2002, The lipofuscin component A2E selectively inhibits phagolysosomal degradation of photoreceptor phospholipid by the retinal pigment epithelium, Proc. Natl. Acad. Sci. USA 99(6):3842–47.

Finnemann, S. C., Bonilha, V. L., Marmorstein, A. D., and Rodriguez-Boulan, E., 1997, Phagocytosis of rod outer segments by retinal pigment epithelial cells requires alpha(v)beta5 integrin for binding but not for internalization, Proc. Natl. Acad. Sci. USA 25(24):12932–37.

Gal, A., Li, Y., Thompson, D. A., Weir, J., Orth, U., Jacobson, S. G., Apfelstedt-Sylla, E., and Vollrath, D., 2000, Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa. Nat. Genet. 26(3):270–71.

Gibbs, D., Kitamoto, J., and Williams, D. S., 2003, Myosin VIIa in RPE phagocytosis and a potential cause of blindness in Usher syndrome 1B, Proc. Natl. Acad. Sci. USA 100(11):6481–86.

Herman, K. G., and Steinberg, R. H., 1982a, Phagosome degradation in the tapetal retinal pigment epithelium of the opossum, Invest. Ophthalmol. Vis. Sci. 23:291–304.

Herman, K. G., and Steinberg, R. H., 1982b, Phagosome movement and the diurnal pattern of phagocytosis in the tapetal retinal pigment epithelium of the opossum, Invest. Ophthalmol. Vis. Sci. 23:277–290.

Karan, G. , Lillo, C., Yang, Z., Cameron, D. J., Locke, K. G., Zhao, Y., Thirumalaichary, S., Li, C., Birch, D. G., Vollmer-Snarr, H. R., Williams, D. S., and Zhang, K., 2005, Lipofuscin accumulation, abnormal electrophysiology, and photoreceptor degeneration in mutant ELOVL4 transgenic mice: a model for macular degeneration, Proc. Natl. Acad. Sci. 102, 4164–69

Liu X., Udovichenko I. P., Brown, S. D., Steel, K. P., and Williams, D. S., 1999, Myosin VIIa participates in opsin transport through the photoreceptor cilium, J. Neurosci. 19:6267–74.

Rakoczy, P. E., Zhang, D., Robertson, T., Barnett, N. L., Papdimitriou, J., Constable, I. J., and Lai, C. M., 2002, Progressive age-related changes similar to age-related macular degeneration in a transgenic mouse model, Am. J. Pathol. 161:1515–24.

Ryeom, S. W., Sparrow, J. R, and Silverstein, R. L., 1996, CD36 participates in the phagocytosis of rod outer segments by retinal pigment epithelium, J. Cell Sci. 109:387–95.

Tsang, S. H., Burns, M. E., Calvert, P. D., Gouras, P., Baylor, D. A., Goff, S. P., and Arshavsky, V. Y., 1998, Role for the target enzyme in deactivation of photoreceptor G protein in vivo, Science 282:117–21.

Vollrath, D., Feng, W., Duncan, J. L., Yasumura, D., D’Cruz, P. M., Chappelow, A., Matthes, M. T., Kay, M. A., and LaVail, M. M., 2001, Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk, Proc. Natl. Acad. Sci. USA 98(22):12584–89.

Young, R. W., 1967, The renewal of photoreceptor cell outer segments, J. Cell Biol. 33:61–72. Young, R. W., 1971, Shedding of discs from rod outer segments in the rhesus monkey, J. Ultra-

struct. Res. 34(1):190–203.

Functional Expression of Cone Cyclic

Nucleotide-Gated Channel in Cone

Photoreceptor-Derived 661W Cells

J. Browning Fitzgerald, Anna P. Malykhina, Muayyad R. Al-Ubaidi, and Xi-Qin Ding

1 Introduction

Cone phototransduction mediated by cone cyclic nucleotide-gated (CNG) channel activation is essential for central and color vision. Rod or cone CNG channels are composed of two structurally related subunit types: the A and the B subunits. The stoichiometry of the rod CNG channel complex has been established by biochemical, biophysical and molecular biological means, showing a heterotetrameric complex composed of three A and one B subunits (Shammat and Gordon, 1999; Zheng et al., 2002). Naturally occurring mutations in the cone channel have been shown to affect cone function and to associate with varieties of cone diseases (Kaupp and Seifert, 2002). Unlike the rod channel, the biochemical properties and functional regulation of the cone CNG channel are much less understood; only recently has the molecular identity of the channel B subunit been revealed (Gerstner et al., 2000). This is primarily due to the difficulty of investigating the cone system in a mammalian retina and lack of an appropriate cone-specific cell line. The recently characterized cone-derived 661 W cell line has been shown to be a useful model to study cone-specific proteins and cone function. The 661 W cell line was originally cloned from retinal tumors of a transgenic mouse line expressing SV-40 T antigen under control of the human interphotoreceptor retinoid binding protein (hIRBP) promoter (Tan et al., 2004). These cells demonstrate cellular and biochemical characteristics of cone photoreceptors and resemble neuronal cells in morphology. They grow as a monolayer, exhibit processes characteristic of neuronal cells and express several markers of cone photoreceptors, including blue and green opsins, transducin and cone arrestin (Tan et al., 2004). However, they do not express rod opsin or arrestin, or rod and cone-specific proteins, such as phosducin, peripherin/rds and rom-1, or the retinal pigment epithelial cell specific protein, RPE65 (Tan et al., 2004). Moreover, these cells have been shown to respond to light stress, oxidative stress and other

X.-Q. Ding

Departments of Cell Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., Oklahoma City, OK 73104, USA, Tel: 405-271-8001 x47959, Fax: 405-271-3548 e-mail: xi-qin-ding@ouhsc.edu

CNGA3-HA was produced by inserting the coding sequence in-frame into the carboxyl terminus of CNGA3 using the quick-change site-directed mutagenesis strategy. The mouse CNGB3 construct was generously provided by Dr. Martin Biel (Ludwig-Maximilians-Universitat Munchen, Germany).

Cells were routinely cultured in Dubelcco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37◦C in a humidified atmosphere with 5% CO2. Transfection was performed using nucleofector technology which is a highly efficient gene transfer method for hard-to-transfect cell lines and for most primary cells (Iversen et al., 2005; Leclere et al., 2005).

2.4 Western Blotting and ICC

Western blotting and ICC were carried out as previously described (Ding et al., 2004; Tan et al., 2004).

2.5 Electrophysiological Recordings

Standard whole-cell patch clamp recordings were performed as previously described (Malykhina et al., 2006) on transfected cells grown on 35 mm culture dishes. For voltage clamp experiments the external solution contained (in mM): NaCl 135, KCl 5.4, NaH2PO4 0.33, MgCl2 1, CaCl2 2, HEPES 5, D-glucose 5.5, adjusted with NaOH to pH 7.4. Pipette solution consisted of (in mM): K+aspartate 100, KCl 30, NaCl 5, MgCl2 2, Na-ATP 2, EGTA 1, HEPES 5 with pH 7.2 adjusted with KOH. Patch electrodes had resistances of 3-5 MΩ when filled with internal solution. Whole cell currents were recorded by using voltage clamp, ramp, and gap-free protocols. For voltage clamp protocol cells were held at –70 mV and series of voltage steps from –70 mV up to +60 mV were applied with 10 mV increments. For ramp protocol cells were held at –50 mV and voltage changes were used in a ramp manner from –80 mV to +80 mV. For gap-free protocol the holding potential was also – 50 mV. All experiments were performed at room temperature (23◦C) and recorded using an Axopatch 200B amplifier (Axon Instrument, Foster City, CA). pCLAMP software (Axon Instruments) was used for data acquisition and analysis.

3 Results

3.1 Expression of CNGA3 and CNGB3 in 661 W cells

RT-PCR was performed to determine whether there are endogenously expressed cone CNG channel subunits in 661 W cells. With primers amplifying the full-length mouse CNGA3, a single band was detected from the wild type and cone-dominant Nrl−/− retinas (generously provided by Dr. Anand Swaroop at the University of

Fig. 1 Expression of

CNGA3, CNGB3 and red/green opsin in 661 W cells and mouse retinas (A–B). RT-PCR detection of CNGA3 (A) and CNGB3 (B). HPRT was used as internal control (C–D). Western blot detection of CNGB3 (C) and green opsin (D) in 661 W cells

Michigan) but no signal was seen in 661 W cells (Fig. 1A). RT-PCR with primers amplifying the full-length mouse CNGB3 showed a weak band in 661 W cells, compared with the ample signal in the mouse retina (Fig. 1B). Consistent with the RT-PCR results, a weak expression of CNGB3 protein in 661 W cells was detected by Western blotting using the anti-CNGB3 antibody (Fig. 1C). With this antibody a single band that migrated at position of 75 kDa was detected in 661 W cells and in mouse retinas. The signal was more abundant in Nrl−/− retina than that in the WT. No CNGA3 was detected with the respective antibody (data not shown) which is consistent with the RT-PCR finding. Expression of green opsin in 661 W cells was detected by Western blotting (Fig. 1D). Thus, these results suggest that there is no detectable, endogenously expressed CNGA3 but a weak expression of CNGB3 in 661 W cells.

3.2Heterologous Expression of Cone CNG Channel Subunits in 661 W Cells

To express CNGA3 in 661 W cells several transfection strategies were tested and only the nucleofector method showed to be effective in introducing the cDNA and expression. Figure 2 shows heterologous expression of CNGA3 and CNGA3-HA in 661 W cells 48 hours post-transfection detected by Western blotting (Fig. 2A) and ICC (Fig. 2B). Expression of the CNGA3 constructs was detected by both antibodies against CNGA3 or HA epitope. A single band migrated at 70 kD was detected in the protein extracts from transfected cells. The aggregates on top of the lanes may reflect an incomplete solubilization or reduction. In ICC examination, the channel

in a mammalian retina. There have been some studies using Xenopus oocytes or HEK293 cells as cellular models to investigate cone CNG channel but many of the results developed from these studies appeared controversial. Studies from Zhong et al using HEK293 cells proposed a 3A:1B stoichiometry of the cone channel complex (Zhong et al., 2002) while a 2A:2B stoichiometry was proposed by Peng et al from studies using Xenopus oocytes (Peng et al., 2004). Similarly, functional importance of calmodulin binding to the cone CNG channel was documented in studies using Xenopus oocytes (Peng et al., 2003) while the irrelevance of calmodulin to the cone CNG channel regulation was proposed by Muller et al from studies using HEK293 cells (Muller et al., 2001). CNG channel is an important component in the phototransduction cascade. The nature that 661 W cells possess includes exhibiting characteristic of neuronal cells and expressing cone specific proteins that are involved in photoactivation and phototransduction (cone opsins, transducin and arrestin, etc) (Tan et al., 2004) provides an ideal internal environment to study the functional regulation and biochemical properties of cone CNG channel. This work was designed to establish the potential use of the cone-derived photoreceptor cell line, the 661 W cell line, as a valuable cellular model to study cone CNG channel. The channel A and B subunits were successfully expressed in these cells and their response to cGMP stimulation was demonstrated by electrophysiological recording. Although this cell line is known to be difficult for transfection, the novel DNA nucleofector technology has proven to be effective in this matter. 661 W cell line stably expressing Bcl2 has been generated and used in a number of studies investigating apoptosis in photoreceptors (Crawford et al., 2001; Kanan et al., 2007). The availability of an in vitro model of cone photoreceptor that closely resembles the native setting will facilitate studies of the cone channel and help understand its physiology and any mutation-related pathology.

Acknowledgments This work was supported by the National Institutes of Health (NIH) COBRE grant P20 RR017703, National Eye Institute (NEI) grant EY14052 and a research grant from American Health Assistance Foundation (AHAF). We thank Dr. Muna Naash’s support in providing experimental facilities.

References

Castagnet P, Mavlyutov T, Cai Y, Zhong F, Ferreira P (2003) RPGRIP1s with distinct neuronal localization and biochemical properties associate selectively with RanBP2 in amacrine neurons.

Hum Mol Genet 12:1847–1863.

Crawford MJ, Krishnamoorthy RR, Rudick VL, Collier RJ, Kapin M, Aggarwal BB, Al-Ubaidi MR, Agarwal N (2001) Bcl-2 overexpression protects photooxidative stress-induced apoptosis of photoreceptor cells via NF-kappaB preservation. Biochem Biophys Res Commun 281: 1304–1312.

Ding XQ, Nour M, Ritter LM, Goldberg AF, Fliesler SJ, Naash MI (2004) The R172W mutation in peripherin/rds causes a cone-rod dystrophy in transgenic mice. Hum Mol Genet 13:2075–2087.

Gerstner A, Zong X, Hofmann F, Biel M (2000) Molecular cloning and functional characterization of a new modulatory cyclic nucleotide-gated channel subunit from mouse retina. J Neurosci 20:1324–1332.