Experimental Eye Research 83 (2006) 380e389 www.elsevier.com/locate/yexer
Multiple, parallel cellular suicide mechanisms participate in photoreceptor cell death* Heather R. Lohr a,1, Kannan Kuntchithapautham a,1, Ashish K. Sharma a,2, Ba¨rbel Rohrer a,b,* a
Department of Neurosciences, Division of Research, Medical University of South Carolina, 173 Ashley Avenue, BSB403, Charleston, SC 29425, USA b Department of Ophthalmology, Medical University of South Carolina, 167 Ashley Avenue, SEI511, Charleston, SC 29425, USA Received 15 November 2005; accepted in revised form 5 January 2006 Available online 19 April 2006
Abstract Photoreceptor degeneration in human photoreceptor dystrophies and in the relevant animal models has been thought to be executed by one common mechanism e caspase-mediated apoptosis. However, recent experiments have challenged this concept. In previous experiments, analyzing gene expression in the degenerating rd/rd mouse retina, we have suggested that the gene defect leads to oxidative stress and altered metabolism, which may induce caspase-dependent and caspase-independent cell death mechanisms such as the activation of cystein-proteases, lysosomal proteases, autophagy and complement-mediated lysis. In this study we asked two questions. First, whether a temporal analysis of these different mechanisms during the course of degeneration would enable us to establish a causal relationship between these events; and second, whether photoreceptor degeneration in different models of photoreceptor dystrophies occurs by activating the same mechanisms. Three models of photoreceptor degeneration were chosen in which photoreceptor degeneration is caused by different events: the rd/rd mouse (calcium overload); the rds/rds mouse (structural defect); and light-damage (LD; oxidative stress). Marker genes were selected for the identified processes. PCR-analysis on laser capture microdissection samples was used to verify the expression of these genes in the rod photoreceptor layer. A temporal relationship between the processes was established at the mRNA level, using quantitative RT-PCR. The time course of gene expression was compared to that of cell loss (loss of rows of photoreceptor nuclei) and apoptosis (TUNEL labeling). Apoptosis and autophagy was analyzed using enzymatic assays. The time course of apoptosis and TUNEL labeling coincide in all three models. Complement-activated lysis was found to either parallel (rd/rd and rds/rds) or precede (LD) the development of TUNEL-positive cells. Autophagy was determined to parallel (rd/rd and LD) or lag (rds/rds) behind the development of TUNEL-positive cells. In all three models, glucose metabolism was found to be increased significantly prior to the onset of cell death, but then dropped in parallel with the loss of cells. The presence of the marker genes was verified by laser capture microdissection, and apoptosis (caspase activity) and autophagy (lysozyme and cathepsin activity) were verified in retina extracts. These results provide evidence that irrespective of whether photoreceptor degeneration is triggered by gene defects (lack of b-PDE or rds/peripherin) or environmental stress (light-damage), a number of pro-apoptotic mechanisms are triggered leading to the degeneration of the photoreceptor cells. The temporal pattern of the different pathways suggests that the non-caspase-dependent mechanisms may actively participate in the demise of the photoreceptors, rather than represent a passive response of the retina to the presence of dying cells. Thus, unless the common upstream initiator for a given photoreceptor dystrophy is found, multiple rescue paradigms need to be used to target all active pathways. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: photoreceptor degeneration; apoptosis; autophagy; complement; metabolism
* Funding information: Funding was provided by NIH grants EY-13520 and Vision Core Grant EY-14793; a postdoctoral fellowship grant for AKS from Fight for Sight, New York, NY; the Kirchgessner Foundation, and an unrestricted grant to MUSC from Research to Prevent Blindness, Inc., New York, NY. BR is a Research to Prevent Blindness Olga Weiss Scholar. * Corresponding author. Department of Neurosciences, Division of Research, Medical University of South Carolina, 173 Ashley Avenue, BSB403, Charleston, SC 29425, USA Tel.: þ1 843 792 5086; fax: þ1 843 792 1723. E-mail address:
[email protected] (B. Rohrer). 1 These authors have contributed equally to this manuscript. 2 Present address: Department of Cell Biology, University of Virginia, PO Box 800732, Charlottesville, VA 22908, USA.
0014-4835/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2006.01.014
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1. Introduction Retinal dystrophies to date are caused by mutations in 158 different genes (RetNet at http://www.sph.uth.tmc.edu/Retnet) and various environmental factors. In the majority of these dystrophies photoreceptor cell death results from a caspasedependent apoptotic mechanism. However, recent evidence suggests that photoreceptor cell death may involve other death pathways such as calpain-mediated cell death, autophagy, proteasome activity and complement-mediated lysis (Rohrer et al., 2004; Sharma and Rohrer, 2004). Retinal photoreceptors are very stable, but at the same time extremely fragile cells (Stone et al., 1999). What makes them extremely fragile is their specialization: the absorption of light and the energy requirements to carry out this task. Although the photoreceptors are equipped to deal with short periods of stress (Stone et al., 1999), any long-term perturbation in either components of the signal transduction cascade, energy metabolism, or structural integrity within the photoreceptors or their support cells (i.e., retinal pigment epithelium or Mu¨ller cells) increases the risk of photoreceptor cell death (Pierce, 2001). Photoreceptor cell death during early postnatal development is a normal process. In the mouse retina, cell death in the outer nuclear layer peaks around postnatal day 7 (P7) and P8 and is complete by P14 (Young, 1984). This early cell death is due to apoptosis (Portera-Cailliau et al., 1994; Young, 1984), a highly regulated and active mode of cell death. Apoptosis is an energy dependent process and involves the activation of effectors such as the caspase proteases (Nicholson and Thornberry, 1997), which ultimately lead to nucleosomal DNA fragmentation and cell death. However, apoptosis is also a physiological strategy to remove damaged cells (Liang and Fesik, 1997), a process that has been proposed to participate in mouse models of retinitis pigmentosa (e.g., Fox et al., 1999; Hao et al., 2002; He et al., 2000; Portera-Cailliau et al., 1994). Autophagy is an additional process by which a cell compartmentalizes and digests itself, providing a mechanism by which cell death can be induced in damaged cells, and is presumed to be a mechanism by which photoreceptors remove outer segments during lightdamage (Reme et al., 1999). Pathologic neuronal degeneration also involves necrosis, which is in response to major insults that lead to cell rupture and release of cellular content into the environment, damaging surrounding cells and leading to inflammatory responses (Nicotera et al., 1999). A strong case for inflammation as part of the causal chain in neurodegeneration has been made for Alzheimer’s disease (Akiyama et al., 2000), age-related macular degeneration (Zarbin, 2004), and the rd/rd and the LD mouse model of retinal degeneration (Rohrer et al., 2004; Zhang et al., 2005). In addition, the released photoreceptor-specific proteins have been shown to be potent immunogens (Adamus et al., 1994). Thus, the interplay between exogenous and intrinsic stressors and survival factors, the cells ability to remove stressors (e.g., degradation of damaged proteins, removal of metabolites, etc.), and the activation of apoptotic and catastrophic death-pathways need to be investigated.
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To investigate the underlying events that can lead to retinal degeneration, we compared three models of photoreceptor degeneration: the rd/rd mouse (Farber, 1995), the rds/rds mouse (Travis et al., 1989), and the light-damage model in albino mice (reviewed in (Penn and Anderson, 1992)). In the rd/rd mouse, the mutation in the b-subunit of the cGMP phosphodiesterase (PDE) results in high levels of cGMP (Farber and Lolley, 1974), which leaves an increased number of the cGMP-gated channels in the open state, thus allowing intracellular calcium (Ca2þ) to rise to toxic levels (He et al., 2000). In the rds/rds mouse, rod outer segments fail to form (Cohen, 1983; Sanyal et al., 1980; van Nie et al., 1978), which is followed by the loss of cell bodies of both rods and cones. Finally, cell death due to light-damage is thought to be due to oxidative stress induced by a prolonged increase in oxygen tension and photo-oxidation (reviewed in (Penn and Anderson, 1992)). In each model, gene expression was analyzed during the known time course of photoreceptor degeneration, using quantitative RT-PCR (QRT-PCR). Marker genes for apoptosis, autophagy, oxidative stress, neuroinflammation and complement activation, and glucose metabolism were selected based on published changes in gene expression obtained from the rd/ rd mouse (Rohrer et al., 2004). The following marker genes were examined: apoptosis e caspase 1 and BIRC4; lysosomal protease activity/autophagy e cathepsin S and lysozyme; oxidative stress e ceruloplasmin and clusterin; neuroinflammation e glial fibrillary acidic protein (GFAP); complementactivated lysis e complement component 1qb (C1qb); and metabolism/glycolysis e 6-phospho-fructo-kinase (6-PFK)). The identified temporal gene expression patterns were compared to cell loss (as shown by the number of cells in the outer nuclear layer (ONL)) and apoptosis (number of TUNELpositive cells in the ONL). Establishment of the temporal relationship between the identified processes suggests that the different mechanisms identified contribute differentially to the early versus the late phase of cell death, depending on the photoreceptor dystrophy. In the rd/rd mouse, apoptosis, autophagy and complement activation are triggered in parallel; in the rds/rds mouse, apoptosis and complement activation precede autophagy; and in the light-damage mouse model, complement activation precedes apoptosis and autophagy. The activity of key enzymes involved in apoptosis (caspase 3 activity) and autophagy (lysozyme and cathepsin activity) were verified in retina extracts using enzymatic assays. The identification of key regulators in neurodegeneration, especially in the context of known human retinal degenerative disorders, will be useful for developing novel therapeutic strategies, which, according to our findings, may require multiple rescue strategies. 2. Material and methods 2.1. Animals 2.1.1. Animal models The rd/rd and rds/rds mice were gifts from Drs. Debora Farber and Gabriel Travis (both at the University of California,
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Los Angeles, CA), respectively. Both are used as the homozygous form of the mutation and are maintained on a C57BL/6 background. C57BL/6 and Balb/c mice were generated from breeding pairs obtained from Harlan Laboratories (Indianapolis, IN). Mice were housed in the Medical University Animal Care Facility under a 12:12 h light:dark cycle with access to food and water ad libitum. The ambient light intensity at the eye level of the mice was 85 18 lux. Based on the known time course of photoreceptor degeneration in the three models (e.g., LaVail et al., 1999; Portera-Cailliau et al., 1994), 8e9 time-points were picked for each animal model. All experiments were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the University Animal Care and Use Committee. 2.1.2. Light exposure Light-exposure experiments were performed on 3-monthold Balb/c mice. Light-exposure consisted of constant fluorescent illumination of w150e175 ft-c for 10 days as described previously (Faktorovich et al., 1992; Rohrer et al., 2003), ensuring that all cages were equidistant to the light source. Littermates were kept under the normal cyclic light conditions for controls. 2.2. Real-time PCR 2.2.1. RNA preparation Age-matched wild type (C57BL/6 and Balb/c) and experimental animals (rd/rd, rds/rds and light-damage (LD)) were sacrificed by decapitation and retinas were quickly isolated and stored in RNA-later (Ambion, Austin, TX) at 20 C (n ¼ 4 per age and time point). Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA), followed by a clean-up
using RNeasy minicolumns (Qiagen, Valencia, CA). The quality of the RNA was examined by gel electrophoresis and spectrophotometry, accepting a 260/280 ratio of 1.95e2.1, as reported previously (Rohrer et al., 2004). 2.2.2. Whole retinas Real-time PCR was performed as published previously (Rohrer et al., 2004). RNA (2 mg each) was used to generate first strand cDNA in reverse-transcription reactions (Invitrogen, Carlsbad, CA). PCR amplifications were conducted using the QuantiTect Syber Green PCR Kit (Qiagen, Valencia, CA) with 0.01 U/mL AmpEraseÒ UNG enzyme (Applied Biosystems, Foster City, CA) to prevent carryover contamination (see Table 1 for primer sequences, gene accession number, expected length, gene ontology and references to document the importance of the particular gene product to the process it is meant to represent). Real-time PCR was performed in triplicate in a GeneAmpÒ 5700 Sequence Detection System (Applied Biosystems, Foster City, CA) using the following cycling conditions: 50 C for 2 min, 94 C for 15 min, 40 cycles of 94 C for 15 s and 58 C for 1 min. Quantitative values were obtained using the cycle number (Ct value), which is inversely proportional to the amount of a specific mRNA species in the tissue sample. Relative gene expression levels were calculated using the equation y ¼ (1 þ AE)DDCt, where AE is the amplification efficiency of the target gene (set at 1.0 for all calculations), and DDCt is the difference between the mean experimental and control DCt values. The DCt value is the difference between the Ct value for a retina-associated gene and the b-actin internal reference control gene (Mitas et al., 2001). 2.2.3. Laser capture microdissection (LCM) Age-matched wild type and experimental animals were sacrificed as described above. Eyes were quickly enucleated,
Table 1 Primer sequences used in quantitative real-time PCR Name
Accession #
Sequence (50 -30 ) forward
Sequence (50 -30 ) reverse
Size
b-actin
NM_007393 NM_009688
Caspase 1
NM_009807
Cathepsin S
NM_021281
Lysozyme
NM_017372
C1qb
NM_009777
GFAP
NM_010277
Ceruloplasmin
NM_007752
Clusterin
NM_013492
6-PFK
NM_008825
TCT CCA GGG AGG AAG AGG AT TGC TCC CGG ATC TTT GGA T GGT CCC ACA TAT TCC CTC CT CAC AGC ACT GAA AGC CCA ACA TGA TAA CAG GCT CAT CTG TCT CA TGG AGA AAA CCT AGA AGC AGC A CCT TCT GAC ACG GAT TTG GT GCA ACC CAG CTT TCA GAT GGT GGA CTG TTC C GT GTC AAC TGA A TTC TTG CAT GTC CAG AGC AC
123
BIRC4
GCT ACA GCT TCA CCA CCA CA GCT GAA AAA ACA CCA CCG CTA A CAC AGC TCT GGA GAT GGT GA TCT AAT CGG ACA TTG CCT GAC A CCA GTG TCA CGA GGC ATT TA CAC CAG GAT TCC ATA CAC AGG A ACA TCG AGA TCG CCA CCT AC ATA ATC AAC CTG TTC CCT GCC A GCC TTG GCA AGA GAT AAG CAT T GGA ACA GCT TTA CGC CTC TG
Gene ontology
References
142
Apoptosis
(Deveraux et al., 1997)
121
Apoptosis
(Enari et al., 1995)
105
Autophagy
105
Autophagy
103 128
Complementactivated lysis Inflammation
(Klionsky and Emr, 2000; Levine and Klionsky, 2004) (Klionsky and Emr, 2000; Levine and Klionsky, 2004) (Morgan and Meri, 1994)
109
Oxidative stress
107
Oxidative stress
117
Glycolysis
(Pannicke et al., 2005) (Arnaud et al., 1988; Osaki et al., 1966) (Kalka et al., 2000) (Rider et al., 2004; Winkler, 1981)
The marker genes to be analyzed were chosen based on the gene expression pattern in the rd/rd mouse published previously (Rohrer et al., 2004).
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anterior chambers and lenses removed, eyecups bisected and frozen in OCT (n ¼ 2 per genotype). Fresh-frozen sections were cut at 15 mm, mounted onto poly-L-lysine coated slides and stored at 80 C. Slides were thawed at room temperature for 30 s and fixed for 30 s in 75% ethanol. OCT was removed by immersion in distilled water 2 times for 30 s each. Tissue sections were then quickly dehydrated by a series of 30-s immersions in 75%, 95% and 100% ethanol followed by xylene for 5 min according to the Arcturus Bioscience, Inc., protocol (Mountainview, CA). Nuclei from the ONL were picked using a PixCell II LCM system (Arcturus Bioscience, Inc.), with a spot size ranging from 7.5 to 15 mm, depending on the size of the ONL, avoiding contamination from neighboring layers (Harada et al., 2000). Nuclei from 2e4 retina sections were collected per sample. Total RNA was extracted from the captured cells by using the Picopure RNA Isolation kit (Arcturus Bioscience, Inc.). T7-based RNA amplification was carried out by using the RiboAmp kit (Arcturus Bioscience, Inc.) and cDNA was synthesized using the Sensiscript RT kit (Qiagen, Valencia, CA), both according to manufacturers’ instructions. This procedure yielded cDNA equivalent to approximately 500 pg of RNA of starting material as judged by comparing Ct values for b-actin of LCM samples with those of samples with measurable RNA concentrations. Only samples that were negative for GFAP were used, to ensure the absence of cross contamination with cells from the inner retina. Quantitative real-time PCR was performed as described above. 2.3. Histology 2.3.1. Eye morphology Paraffin sections were collected and processed for morphological analysis as described before (Rohrer et al., 1999). In short, animals were decapitated, and eyes were enucleated and immersion-fixed in Carnoy’s fixative for 2 h. Eyes were dehydrated and embedded in paraffin in transverse orientation. Seven micrometer sections of the eye were cut in dorsoventral orientation and dried onto poly-L-lysine-coated glass slides. Sections were dewaxed and rehydrated through xylene and a graded series of ethanol, stained with toluidine blue solution and coverslipped using DPX medium. The number of rows of photoreceptor nuclei were counted in the central retina (superior and inferior, within 100 mm of the optic nerve head) as described previously (Rohrer et al., 2003). Two to three measurements were made per field, which were averaged to provide a single value for the entire retina. Each group consisted of 4e7 animals. 2.3.2. TUNEL TUNEL labeling was performed according to the protocol provided by the manufacturer (Roche Diagnostics, Indianapolis, IN). In short, eyes were fixed in 2% paraformaldehyde for 2 h at 4 C followed by dehydration and paraffin embedding as described above. TUNEL labeling (TdT-mediated dUTP nickend labeling) was performed on 7 mm paraffin sections, and the DNA strand breaks were labeled with fluorescein for
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visualization. Slides were examined using a fluorescein isothiocyanate (FITC) filter on a Zeiss microscope equipped with fluorescence. The number of TUNEL-positive nuclei per 200 mm window was counted in the central retina (superior and inferior, within 100 mm of the optic nerve head) on 4e6 sections per animal. These measurements were averaged to provide a single value for the entire retina. Each group consisted of 2e3 animals. 2.4. Enzymatic assays Enzymatic activity was measured using appropriate fluorescent substrates provided in the enzyme activity assay kits in retina homogenates (caspase 3 and lysozyme, Molecular Probes, Eugene, OR; cathepsin B, Calbiochem, San Diego, CA) as described previously (Sharma and Rohrer, 2004). To analyze activity in whole retinas, retinas were dissected, pooling two retinas per sample, the tissue resuspended in 100 mL lysis buffer, kept on ice at 4 C for 15 min, and then homogenized by tissue grinding and sonication on ice. The supernatant was collected after centrifugation at 20,000 g for 15 min, transferred to a 96-well plate to which 10 mL of 10X reaction buffer and 5 mL of substrate were added. After appropriate incubation times, the samples were read in a fluorometer equipped with appropriate excitation and emission filters. Protein content was measured using the Bradford Folins reagent method (Biorad Laboratories, Hercules, CA) and enzyme activities were expressed as relative fluorescence units (RFU) per milligram protein of each sample. The arbitrary values were presented as the mean SD of three to five samples. 3. Results 3.1. Anatomical analysis To examine whether the temporal relationship between the multiple degenerative processes is comparable in unrelated models of photoreceptor dystrophy, marker gene expression was correlated with morphological endpoints, the thickness of the outer nuclear layer, and TUNEL staining. The morphologic data for both the rd/rd and rds/rds mouse have already been published (Portera-Cailliau et al., 1994) and are replotted here, whereas the anatomical data for the light-damage model was obtained for this publication. In Fig. 1A the temporal relationship of photoreceptor cell loss and TUNEL staining in the three different dystrophies is documented. In the rd/rd mouse the number of TUNEL-positive cells occurs in a single wave, with the number of TUNEL-positive cells peaking at P14 (Fig. 1A, left-hand column). In the rds/rds mouse, TUNEL-positive cells occur in two waves, involving an early wave (peaking at P18) and a late wave (ramping up by P30) (Fig. 1A, middle column). Finally, in the constant lightdamage model, TUNEL-positive cells can be detected within 24 h of light onset, with a continued increase over time (Fig. 1A, right-hand column).
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3.2. Temporal relationship between degenerative processes
that oxidative stress markers are elevated, but is decreased subsequently (Fig. 1F).
Retinal gene expression for specific gene clusters was compared to the known progression of outer nuclear layer loss and TUNEL staining as described above. Genes for each individual cluster included: apoptosis e caspase 1 and BIRC4; lysosomal protease activity/autophagy e cathepsin S and lysozyme; oxidative stress e ceruloplasmin and clusterin; neuroinflammation e glial fibrillary acidic protein (GFAP); complement-activated lysis e complement component 1qb (C1qb); and metabolism/ glycolysis e 6-phospho-fructo-kinase (6-PFK)). First, in the rd/rd mouse retina (Fig. 1, left-hand column), markers for apoptosis (Fig. 1B), autophagy (Fig. 1C) and complement activation (Fig. 1E) were activated in parallel and follow the time course of TUNEL labeling, suggesting that all three mechanisms actively participate in photoreceptor cell death. Please note that BIRC4 expression, an endogenous caspase inhibitor is a mirror-image to that of caspase 1. Oxidative stress markers (Fig. 1D) are constitutively unregulated, suggesting that the lack of b-PDE, which is expressed as early as P2.5 at the mRNA level (Mouse Retina SAGE Library, http://itstgp01.med.harvard.edu/retina/default.asp), leads to early oxidative stress. Finally, message for 6PFK (Fig. 1F) is upregulated until P10, suggesting that the photoreceptors require excess ATP to maintain their membrane potential in the presence of open cGMP-gated channels, whereas expression drops once photoreceptor cell loss commences. Second, in the rds/rds mouse retina (Fig. 1, middle column), markers for apoptosis (Fig. 1B) and complement activation (Fig. 1E) again parallel TUNEL staining, whereas markers for autophagy (Fig. 1C) are induced later, possibly in response to the presence of dying cells. Oxidative stress markers (Fig. 1D) and GFAP, a marker for inflammation or necrosis (Fig. 1E), are elevated to varying degrees throughout the entire time period studied. Again, 6-PFK was found to be increased early, but decreased after postnatal day 14 (Fig. 1F). Third, in the light-damage model (Fig. 1, right-hand column), which is an oxidative stress model, the TUNEL-positive phase of degeneration is characterized by an increase in caspase 1 (Fig. 1B), and the induction of autophagy genes (Fig. 1C). Interestingly, oxidative stress markers (Fig. 1D), complement activation markers, and GFAP levels (Fig. 1E) are upregulated within less then 12 h after the onset of light. Finally, message for 6-PFK is increased during the period
3.3. Verification of outer nuclear layer-specific expression To confirm that the genes identified in the whole retina by QRT-PCR reflect changes in gene expression in the photoreceptors themselves, laser capture microdissection experiments were performed on retinas from all three animal models at the late time point, using all but two primers investigated at the whole retina level. Only one of the lysosomal proteases was investigated, and GFAP due to its’ known expression in glial cells served as a negative control as described in Section 2. Good correlation between the data determined for whole retina and photoreceptors was found (Table 2), supporting the validity of our experiments. For all genes, the trend (up- versus down-regulated) was confirmed, although the absolute values did differ in some cases, suggesting that gene expression changes identified in the retina include both changes in the photoreceptors as well as in the inner retina. 3.4. Enzyme analysis for autophagy and apoptosis For the functional analysis, we focused on one time point during the execution phase of cell death, testing whether enzymes that are known to be indicative of both apoptosis and autophagy are more active in the mutant or damaged retinas. The hallmarks of apoptosis are DNA laddering, externalization of phosphatidylserine and activation of caspases (for review see Adams, 2003; Danial and Korsmeyer, 2004), whereas the activation of proteases such the cathepsins, lysozyme and proteasomal proteins in the autophagosome/autolysosome-like organelles is indicative of autophagy (Klionsky and Emr, 2000; Levine and Klionsky, 2004). Thus, enzymatic assays for caspase 3, which is the main executor caspase, and is activated by caspase 1, cathepsin B and lysozyme were performed on time point during the peak of degeneration in rd/rd (P15), rds/rds (P30), and light-damaged (10 days of light-damage) retinas. In all three sets of retinas, enzymatic activities were increased at least 2-fold (Fig. 2). 4. Discussion Based on recent publications that a model involving only caspase-mediated cell death may not be sufficient to explain
Fig. 1. Temporal analysis of cell loss and marker gene expression. To correlate marker gene expression with cell loss and apoptosis, retinas from the three different models, rd/rd (left-hand column), rds/rds (middle column) and light-damage (right-hand column) were analyzed for morphology (A) and gene expression using quantitative RT-PCR (QRT-PCR) (BeF). A: Photoreceptor cell loss was documented by plotting the thickness of the outer nuclear layer as a percent of the agematched control (red), and the number of TUNEL-positive cells per area of retina (black). Please note that the data for the rd/rd and the rds/rds mouse are replotted from Portera-Caillau and colleagues (Portera-Cailliau et al., 1994), whereas those for the light-damage mouse model were newly obtained, following the same procedures. As shown by Clarke and colleagues (Clarke et al., 2000), cell loss data can be fitted with an exponential, whereas TUNEL-labeling has a unique distribution. Data are expressed as mean values. BeF: The temporal expression of a set of marker genes was examined using QRT-PCR on cDNA generated from whole retina. Candidates were examined from the following categories: (B) apoptosis e caspase 1 and BIRC4; (C) lysosomal protease activity/autophagy e cathepsin S and lysozyme; (D) oxidative stress e ceruloplasmin and clusterin; (E) neuroinflammation e glial fibrillary acidic protein (GFAP) and complement-activated lysis e complement component 1qb (C1qb); and (F) metabolism/glycolysis e 6-phospho-fructo-kinase (6-PFK)). To allow for direct comparison with the morphological correlate of apoptosis (TUNEL; in B-E) or photoreceptor cell loss (F), the relevant trace is replotted in each graph (gray). Data are expressed as mean SD (n ¼ 3).
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Table 2 Comparison of whole retina and ONL nuclei using quantitative RT-PCR P17 rd/rd
Gene investigated
P30 rds/rds
Balb/c 10 day LD
Name
Gene ontology
Retina
ONL
Retina
ONL
Retina
ONL
BIRC4 Caspase 1 C1qb Ceruloplasmin Clusterin Lysozyme 6-PFK
Apoptosis Apoptosis Complement activation Oxidative stress Oxidative stress Autophagy Metabolism
6.66 1.26 3.53 0.65 3.20 0.46 1.92 0.43 2.19 0.48 4.94 0.22 6.66 0.99
4.97 2.09 3.37 1.36 8.58 1.73 1.89 0.80 0.94 0.00 23.03 2.73 12.5 1.50
9.34 1.88 6.08 0.76 4.99 2.21 1.79 0.62 4.49 1.32 18.92 5.83 1.52 0.21
1.34 0.87 11.17 3.33 7.55 1.46 8.56 3.52 2.71 0.94 1.24 0.07 2.44 0.30
3.08 0.39 8.04 2.20 5.14 1.70 1.80 0.09 2.85 0.18 12.60 0.36 4.30 0.0
0.51 0.31 23.53 3.55 4.50 1.57 8.57 0.02 4.25 0.95 4.36 1.18 n.d.
To confirm that the PCR data obtained from the whole retina is a reflection of events in the photoreceptors LCM samples were obtained from the outer nuclear layer (ONL) from P17 rd/rd, P30 rds/rds and 10 day light-damaged retinas. QRT-PCR was performed with a subset of the marker genes selected for the time course analysis, revealing good correlation between the two data sets. Please note that the apparent difference between levels may be due to the fact that for example in a P17 rd/rd retina, the photoreceptors only represent a small portion of the overall number of cells, in comparison to the pure selection of the LCM sample.
photoreceptor cell death in various photoreceptor dystrophies, we set out to further investigate whether and when the caspase-dependent and caspase-independent events occur during the time course of degeneration. We argued that a temporal analysis of these different mechanisms would enable us to establish a causal relationship between these events. In particular, we argued that an event occurring either ahead or in parallel with the presence of TUNEL-positive cells is participating in the demise of the photoreceptors, whereas an event occurring after the peak of TUNEL might be in response to the presence of dying cells. The second question asked whether photoreceptor degeneration in different models of photoreceptor dystrophies share a common repertoire of mechanisms and execute cell death by activating the degenerative mechanisms in a comparable temporal order. The time course data, analyzing gene expression of a number of select marker genes for the identified events, suggests that in all three models, apoptosis and complement activation appear to participate in the elimination of photoreceptor cells. Autophagy on the other hand appears to be involved in photoreceptor degeneration in the rd/rd mouse and during light-damage, whereas in the rds/rds mouse this mechanism appears to be involved in clearing away the photoreceptor cell debris. In addition, it was found that 6-PFK is upregulated early in the process of degeneration, suggesting that additional APT is required to counterbalance the insult (e.g., maintain membrane potential, remove defective outer segments or adjust photoreceptor outer segment lengths), whereas later in degeneration, the drop in 6PFK is either coincidental or precedes photoreceptor cell loss. Although changes in gene expression are a powerful indicator that the activity of the pathways they participate in is altered, it has to be kept in mind that changes in gene expression are neither sufficient nor required for changes in activity. Thus, in follow-up experiments we will analyze the activities of the pathways predicted to be involved, taking our clues from the timing established here. According to a recent review (Pierce, 2001), there are at least four different mechanisms that are involved in photoreceptor degeneration: 1) chronic metabolic overload; 2) disruption of OS disc morphogenesis; 3) dysfunction of the RPE; and 4) chronic activation of the phototransduction cascade. It has
been suggested that apoptosis is the common mediator of cell death under all these conditions (reviewed in Travis, 1998). However, our own experiments (Rohrer et al., 2004; Sharma and Rohrer, 2004) and those of others suggest that other mechanisms such as the activation of calpain (Doonan et al., 2005), the participation of a neuroinflammatory cascade (Zarbin, 2004), or the involvement of autophagy (Guimaraes et al., 2003), may play a role in retinal cell death both during degeneration and development. Thus, the main goal of this study was to further characterize this suggested convergence of cell death mechanisms. For this purpose we chose three models of rod degeneration from three of the four mechanisms indicated above; the rd/rd mouse (mechanism 1), the rds/rds mouse (mechanism 2), and the light-damage model in albino mice (mechanism 4). 4.1. Morphological endpoints of photoreceptor cell loss In order to be able to correlate gene expression with morphological outcomes, cell loss was documented by examining the number of rod nuclei and the presence of TUNEL-positive cells over time. As shown by Clarke and colleagues (Clarke et al., 2000), photoreceptor degeneration in rd/rd, rds/rds and light-induced photoreceptor cell loss can be described by a ‘‘one-hit kinetic model’’, leading to an exponential decline in the number of cells (see also Fig. 1A). In their model, it is presumed that the mutant or light-challenged photoreceptors are in a mutant steady state (MSS). Cell death occurs randomly in cells after the concentration of a presumed pathogenic compound X has reached a threshold (summarized in (Pacione et al., 2003)). Differences in the MSS would produce the different time course of cell loss observed in different models. Based on the time course of TUNEL-staining (Fig. 1A) and the known onset of MSS e in the wild type mouse b-PDE expression starts wP2.5, the peripherin gene is expressed as early as P4.5 (Mouse Retina SAGE Library, http://itstgp01.med.harvard.edu/retina/default.asp) and lightexposure leads to changes in gene expression within the first 24 h (Chen et al., 2003 and Fig. 1, right-hand column), in light-damage the pathogenic compound X appears to reach threshold fastest among the three models studied.
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Fig. 2. Enzyme activity assays. Enzymatic assays were performed on retina extracts from the late stage of degeneration (P17 rd/rd; P30 rds/rds; and 10 days of light-damage). In all three sets of retinas, apoptosis mediated by caspase 3 and autophagy mediated by cathepsin B and lysozyme was confirmed. Enzyme activities were expressed in relative fluorescence units (RFU) per milligram of protein per each sample. Data are expressed as mean SD (n ¼ 3).
4.2. Common mechanisms of photoreceptor degeneration Eight or nine time points in the progression of degeneration were picked for each model and gene expression was compared using quantitative RT-PCR. The temporal relationship between these processes suggest first, that the upregulation of oxidative stress genes as well as an increase in glycolysis are early markers of photoreceptor degeneration; and second, that autophagy (with the exception of the rds/rds model), complement-mediated lysis and neuroinflammation appear to participate actively in the demise of the photoreceptors, rather than representing a secondary response to the presence of dying photoreceptors. The presence of oxidative stress genes in the three models was to be expected (see below). Altered metabolism has been demonstrated in both the RCS rat (Graymore, 1964) and the rd/rd mouse. In the rd/rd mouse, metabolism, as judged by oxygen consumption, glucose utilization, lactic acid production and LDH5 activity, was increased prior to P12, followed by a rapid decrease (Acosta et al., 2005; Blanks et al., 1974; Noell, 1965). Thus, here we have confirmed the time course of altered metabolism in the rd/rd mouse using a different marker (6-PFK expression, a key enzyme in glucose metabolism), which not only extends the data on altered metabolism in the rd/rd mouse, but also validates its choice as a marker for altered metabolism in the other two models. The presence of markers for apoptosis, autophagy, complement-mediated lysis and neuroinflammation during the presence of TUNEL-labeled cells, or even preceding the phase of TUNEL-labeling, further supports the notion that caspases are no longer the sole executioners of apoptosis (e.g., Doonan et al., 2005; Guicciardi et al., 2000; Wenzel et al., 2005).
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Classically, three different types of cell death have been distinguished: apoptosis, autophagy, and necrosis, as described above. However, the boundaries, in particular between autophagy and apoptosis, have never been clearly defined, due to significant cross talk. For example, cathepsin B has been implicated both in the apoptosis-dependent hepatocyte (Guicciardi et al., 2000), 661W (Sharma and Rohrer, 2004), and rd/ rd photoreceptor cell death (Doonan et al., 2005; Sharma and Rohrer, 2004), as well as in apoptosis-independent photoreceptor cell death (Wenzel et al., 2005). In addition, if one of the two pathways is inhibited (i.e., by pharmacological intervention), the cell can still die by using the other mechanism (Lockshin and Zakeri, 2004). Thus, cell death seems to involve a continuum of mechanisms. The early presence of the complement component C1qb in all three models is a novel finding. The complement system is part of the immune system. It is one of the major means by which the body recognizes foreign antigens and pathogens as well as tissue injury, ischemia, apoptosis, and necrosis (reviewed in (Bohana-Kashtan et al., 2004; Fearon and Locksley, 1996; Holers, 1995)). The end result of the activation of the complement cascade is dependent upon the condition of activation, whether lytic or sublytic. The main activity appears to be lytic activation leading to the lysis of cells through the generation of the membrane attack complex. However, sublytic activation of the complement cascade can lead to the recruitment of inflammatory cells, the generation of proinflammatory, pro-thrombotic or pro-fibrotic mediators, the enhanced activation of apoptosis and the clearance of apoptotic cells by C1q interacting with C1q receptors. Finally, sublytic activation may also exert anti-apoptotic effects by making cells more resistant to future lytic complement. Without a more detailed analysis of the various complementinduced signaling pathways, it is however not possible to predict the role of the complement system in mouse photoreceptor degeneration. The finding is however particularly interesting in light of the recent findings that mutations in the complement component, Factor H, are a key factor in agerelated macular degeneration (Edwards et al., 2005; Haines et al., 2005; Klein et al., 2005). 4.3. Why caspase-dependent mechanisms aren’t enough Although there is clear evidence that apoptotic mechanisms are involved in photoreceptor cell death (reviewed in Travis, 1998), they seem not to be sufficient to account for all the cell loss occurring in the mouse models of photoreceptor dystrophies. Yoshizawa and coworkers (Yoshizawa et al., 2002) demonstrated that caspase-3 inhibitors only delayed photoreceptor degeneration in C3H mice carrying the rd gene. Likewise, the over-expression of Bcl-2 in photoreceptors did not prevent or ameliorate photoreceptor degeneration (Joseph and Li, 1996). This in-vivo data is consistent with our findings in a cell culture model of the rd/rd photoreceptor (calciumionophore stimulated 661W photoreceptors) that shows that cell death is only transiently inhibited by the caspase 3 inhibitor (Sharma and Rohrer, 2004).
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The requirement for additional mechanisms other then caspase-mediated apoptosis in the demise of the rds/rds photoreceptors was also suggested by experiments demonstrating that although these photoreceptors express caspase 3 (Hughes et al., 2004) (see Table 2), their degeneration can only be delayed, but not prevented by Bcl-2 over-expression (Nir et al., 2000). The presence of caspase-mediated mechanisms in lightinduced photoreceptor degeneration has been unclear. It is agreed that rod degeneration in response to continuous light-exposure appears to involve apoptosis (i.e., photoreceptors are positive for TUNEL); yet Li and colleagues (Li et al., 2003) reported that caspase-3 is not up-regulated at the protein level and experiments by Donovan and colleagues suggests that caspase3 activity may not be increased during light-damage (Donovan et al., 2001). However, both groups use rhodopsin-independent, bright light-induced photoreceptor degeneration, whereas the results reported here are from rhodopsin-dependent, moderate light-exposed animals, further confirming the difference between these two models (Hao et al., 2002). Taken together, our data suggests that irrespective of which event triggers rod photoreceptor cell death, whether a genetic defect or an environmental insult, the first response represents an upregulation of stress genes and an apparent increase in glucose metabolism, to potentially provide sufficient ATP to maintain cellular homeostasis. After a certain delay, the time which is specific to each photoreceptor dystrophy model, cell death is initiated, which may involve multiple death pathways such as apoptosis, autophagy, and complementmediated lysis. The presence of this complex set of pathways clearly provides a significant challenge to the identification of treatment strategies for the prevention of photoreceptor degeneration. Acknowledgements We thank Drs. Debora Farber and Gabriel Travis for the rd/ rd and rds/rds mice, respectively, Craig Crosson for helpful discussions and Luanna Bartholomew for editorial assistance. We wish to acknowledge use of the Laser Capture Microdissection Facility of the Hollings Cancer Center Tumor Bank, supported by DOE/Office of Science and NIH grant DE-FG02-01ER63121.
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