THE ROLE OF INTERLEUKIN-6 SIGNALLING MOLECULES IN … · ii The role of Interleukin-6 signalling molecules in murine models of inherited photoreceptor degeneration Doctor of Philosophy,
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THE ROLE OF INTERLEUKIN-6 SIGNALLING
MOLECULES IN MURINE MODELS OF INHERITED
PHOTORECEPTOR DEGENERATION
by
Michael Joseph Szego
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Department of Molecular Genetics University of Toronto
1.4.2 Cell death by apoptosis ...................................................................................................................... 17
1.4.3 Common kinetics of cell death ............................................................................................................ 18
1.4.4 Implications of these common features occurring in IPD ...................................................................... 18
1.5 Microarrays and IPD ........................................................................................................................... 19
1.5.1 Identification of photoreceptor-enriched genes ....................................................................................... 19
1.5.2 Identification of regulatory networks in the retina................................................................................ 21
1.5.3 Gene expression changes in response to IPD....................................................................................... 22
1.6.2 Brain-derived neurotrophic factor and nerve growth factor .................................................................... 27
1.6.3 The Interleukin-6 family of cytokines ................................................................................................. 28 1.6.3.1 Pathways activated by IL-6 cytokines................................................................................................................ 29
3.3.2 Histology and outer nuclear layer measurements.................................................................................. 84
3.3.3 Retinal protein isolation..................................................................................................................... 84
3.3.6 In situ hybridization.......................................................................................................................... 88
Table 1.1: Retinal responses to IL-6 cytokine activation .......................................................................... 31
Table 2.1: List of qPCR primers ................................................................................................................... 43
Table 2.2: Comparison of transcript fold-differences as determined by microarray and qPCR
analyses in Rom1-/- and wild-type retinas............................................................................................. 51
Table 2.3: Comparison of transcript fold-differences as determined by microarray and qPCR
analyses in Rds+/- and wild-type retinas ............................................................................................... 55
Table 3.1: List of primers used for qPCR.................................................................................................... 85
Table 3.2: Quantification of selected differentially expressed transcripts in three different mouse
models of IPD........................................................................................................................................ 90
ix
List of Figures
Figure 1.1: Structure of the vertebrate retina. ................................................................................................3
Figure 1.2: Structure of a rod photoreceptor. ................................................................................................4
Figure 2.1: Summary of the Rom1-/- normalized microarray data............................................................. 47
Figure 2.2: Poor concordance between qPCR and microarray analyses in the Rom1-/- model of IPD50
Figure 2.3: Summary of the Rds-+/- normalized microarray data .............................................................. 53
Figure 2.4: Moderate concordance between qPCR and microarray analyses in the Rds+/- model of
transgene (P347S) pERK phosphorylated extracellular signal-regulated kinase PR Photoreceptor pStat-1 phosphorylated signal transducer and activator of transcription-1 pStat-3 phosphorylated signal transducer and activator of transcription-3 qPCR Quantitative polymerase chain reaction rAAV Recombinant adeno-associated virus Rd1 Retinal degeneration-1 Rds Retinal degeneration slow Rom1 Rod outer segment membrane protein-1 RP Retinitis pigmentosa RPE Retinal pigment epithelium Socs-3 Suppressor of cytokine signalling-3 Stat-1 signal transducer and activator of transcription-1 Stat-3 signal transducer and activator of transcription-3 Tyk Tyrosine kinase
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1. Introduction
1.1 Structure of the mammalian retina
The mammalian retina is a highly ordered tissue, composed of four cellular layers
that line the back of the eye (Figure 1.1) (Stryer, 1996). The primary function of the retina is to
convert visual cues into a neural signal, which then travels to the visual cortex for further processing
resulting in sight (Stryer, 1996). As the most accessible organ of the central nervous system, the
retina has been extensively characterized (Hubel, 1995; Masland, 2001). Santiago Ramon y Cajal
published a series of very accurate anatomical drawings of the vertebrate retina in 1900 (Ramón y
Cajal and Bresler, 1900). Since then, the neuroanatomy of the retina has been further refined and the
functional relationship between many of the cell types in the retina have been characterized (Hubel,
1995). In addition, the biochemical basis of vision has been well characterized and many genes
required for normal retinal development to occur have been identified (Molday, 1998; Livesey and
Cepko, 2001). The retina is also a non-essential organ. Mutations that affect retinal function or
viability are compatible with life. Consequently, mutations in virtually any gene necessary for eye
development alone, or for ocular function alone, can occur and be studied. In the context of retinal
degeneration, for example, 193 genes or loci associated with inherited retinal degenerations have
been identified (Daiger et al., 2008), making the retina an ideal system for studying neuropathological
mechanisms.
1.1.1 Neuronal cell types in the retina
Photoreceptors (PRs), which constitute 70% of the cells in the mammalian retina (Young,
1985), absorb light and transduce it into a neural signal. Rod PRs are most sensitive to low intensity
monochromatic light, while cone PRs function predominantly at higher light intensity where they are
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involved in detecting colour (Hubel, 1995). In humans, rods and cones exhibit a non-uniform
distribution in the retina (Curcio et al., 1990). The fovea is a region near the center of the retina that
has a high concentration of cones and is most sensitive to detail. The concentration of cones rapidly
decreases with increasing distance from the fovea, while the concentration of rods increases (Curcio
et al., 1990). Retinas of mice, in contrast, exhibit an almost uniform distribution of rods and cones
(Carter-Dawson and LaVail, 1979). Thus, mice do not have an analogous structure similar to the
fovea.
Rod PRs are polarized neurons that consist of three major compartments: the outer segment
(OS), the inner segment (IS), and the synaptic region (Figure 1.2). The synaptic region is the most
proximal region of the PR and transmits signals to the interneurons of the inner nuclear layer. The
inner segment contains the PR nucleus and most of the cell’s biosynthetic machinery (Kandel, 2008).
Many of the proteins made in the inner segment, are actively transported through a structure called
the connecting cilium to the OS (Kandel, 2008). The OS is a modified cilium, containing about 1000
membranous disks in primates (Young, 1971), stacked in an array oriented perpendicular to the long
axis of the OS. Disk morphogenesis occurs as evaginations of the connecting cilium (Steinberg et al.,
1980). OS disk renewal occurs at a rate of 10% per day in primates (Young, 1971), with the older
disks being phagocytosed by the retinal pigment epithelium at the distal tip of the OS in a process
that is calibrated such that the OSs maintain their overall length and structure (Kandel, 2008). The
high renewal rate may be critical to prevent photodamage from occurring on PR molecules (Young,
1976).
Mature OS disks are covered with rhodopsin and are the site of light absorption and
phototransduction. Rhodopsin, which accounts for greater than 70% of all protein in PRs (Molday,
1998), consists of the protein moiety opsin, bound to the light sensitive chromophore, retinal
(Stryer, 1996). Upon light exposure, 11-cis retinal isomerizes into 11-trans retinal which causes a
conformational change in opsin, resulting in activated transducin, which biochemically amplifies the
signal further; the end result of this process is a drop in cGMP concentration, that results in closed
cGMP-gated Ca2+ channels and a decrease in the release of glutamate from the PR axon terminal
(Molday, 1998; Hargrave, 2001). PRs transmit signals to bipolar cells in the inner nuclear layer (INL),
which in turn send a signal to ganglion cells (Figure 1.1). Axons from the ganglion cells then
converge to form the optic nerve, which sends visual signals into the visual cortex for higher
processing (Hubel, 1995).
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Figure 1.1: Structure of the vertebrate retina. The neural retina has three nuclear layers, each composed of specific cell types. The nuclear layers are separated by the outer and inner plexiform layers, which are composed of synaptic terminals. On the left is a cartoon of the retina with corresponding structures indicated on a toluidine-stained section of a wild-type adult murine retina. A=amacrine cell, B=biplolar cell, C=cone photoreceptor, G=ganglion cell, H=horizontal cell, M=Müller cell, R=Rod photoreceptor.
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Figure 1.2: Structure of a rod photoreceptor. The rod PR is composed of the following structures: the outer segment (OS), the inner segment (IS), the cell body and the synaptic terminal. A single outer segment disk has been enlarged on the right. Three OS disk proteins relevant to the research in this thesis include the photopigment rhodopsin, an intrinsic membrane protein of the disk membrane; Peripherin or Rds, which is localized to the disk rims and forms a mixture of disulfide-linked dimers (indicated), octomers and higher-order oligomers. Rds can also form noncovalent homo- and hetero tetramers with Rom-1, another disk rim protein. Similar to Rds, Rom1 can also form homodimers but is not usually observed in homomeric higher order structures (reference Biochemistry. 2008 Jan 29;47(4):1144-56. and Loewen and Molday 2000).
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Other neuronal cell types in the retina function to modulate the neural signals originating from PRs.
A single horizontal cell is capable of providing feedback to multiple PR cells. Horizontal cells are
thought to enhance the contrast between adjacent light and dark visual fields by allowing the PR at
the center of a visual field to excite the ganglion cell below it, while inhibiting neighboring PRs
(Masland, 2001). In contrast, amacrine cells can modulate the activity of bipolar and ganglion cells
(Kolb et al., 1992; Masland, 2001).
1.1.2 Retinal pigment epithelium
The retinal pigment epithelium (RPE) is a monolayer of cells situated immediately behind
the PRs. The RPE is critical to the function and survival of PRs. For example, if the RPE is
separated from PR layer, the PRs will die (Anderson et al., 1983; Berglin et al., 1997). One of the
main roles of the RPE is in vitamin A metabolism in the retina. As previously discussed, all-trans
retinaldehyde results from light-activated rhodsopsin, which is subsequently isomerized to all-trans
retinol by another enzyme located in the PR OS, and transported to the RPE, where it is converted
back to 11-cis retinaldehyde (Stryer, 1996). The RPE65 gene encodes an isomerase required for this
conversion, and mutations in RPE65 have been associated with PR degeneration (Marlhens et al.,
1997). The RPE is also involved in the phagocytosis of disks at the distal tip of the OS. The RPE is
also thought to provide trophic support to PRs. For example, an in vitro study showed that PR
cultures survived longer in the presence of RPE-conditioned media, suggesting the conditioned
media contained trophic factors that improved PR survival (Gaur et al., 1992).
1.1.3 Müller Glia
Müller cells are radial glia and the principal glial cell type in the vertebrate retina (Bringmann
et al., 2006). Müller glia span from the outer limiting membrane, which is formed by the junction
between Müller cells and PR inner segments to the inner limiting membrane at the base of ganglion
cells, ensheathing all retinal neurons in between (Bringmann et al., 2006). This morphological
relationship allows Müller cells to carry out their many functions. For example, Müller cells play an
important role in neurotransmitter recycling. Glutamate is the dominant excitatory neurotransmitter
in the retina and its timely clearance is required so that excitatory synapses can continue to function
normally; neurotoxicity can also occur in the presence of excessive glutamate (Barnett and Pow,
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2000). Müller cells express the glutamate/aspartate transporter GLAST, which rapidly removes
glutamate from synapses; glutamine synthetase then catalyzes the conversion of glutamate into
glutamine within Müller cells (Derouiche and Rauen, 1995). Finally, glutamine is transported back to
neurons as a precursor required for glutamate synthesis (Bringmann and Reichenbach, 2001).
The maintenance of retinal potassium ion (K+) homeostasis is another critical activity of
Müller cells. Müller cells express a specific class of K+ channel called inward-rectifying K+ channels
which make the cell highly permeable to K+ (Newman, 1993), an ion released by active neurons.
Excess K+ is absorbed by Müller cells from the neuronal extracellular space, and is distributed to the
blood, the vitreous and the subretinal space (Bringmann and Reichenbach, 2001).
Several other important functions of Müller glia have been identified. The retina produces a
lot of water as a result of its high metabolic rate (Bringmann and Reichenbach, 2001). In addition,
water influx also occurs as a result of metabolic substrate uptake (Bringmann and Reichenbach,
2001). Aquaporins present on Müller glia facilitate the redistribution of water out of the inner retinal
tissue (the RPE facilitates the redistribution of water out of the subretinal space) (Bringmann and
Reichenbach, 2001). Müller cells also provide reduced glutathione to neurons, which reduces the
number of free radicals and reactive oxygen species (Schutte and Werner, 1998; Bringmann and
Reichenbach, 2001). Finally, Müller glia can phagocytose debris from dead neurons or RPE cells
(Francke et al., 2001). While Müller glia play a role in maintaining homeostasis in the healthy retina,
there is increasing evidence that they also play a neuroprotective role in retinas under stress (Zack,
2000; Bringmann and Reichenbach, 2001).
Müller glia also appear to be important in PR degeneration (Zack, 2000). PR cell death can
be caused by pathogenic mutations or injury (LaVail et al., 1992; Pacione et al., 2003). Several
Gene expression was quantified by the standard curve method (ABI, 2001). Typically, the
expression of two genes was interrogated in a single 96-well qPCR reaction plate. To compare the
expression level of a gene between samples, primers were used in reactions as previously described:
four standard curve dilutions performed in triplicate, six separate cDNA samples from three mutant
and three wild-type individuals also run in triplicate. A standard curve was performed for each
reaction as recommended for accurate quantification (Bustin, 2002). Additionally, three ddH2O
controls and one –RT control for each sample is also present on each plate. Typically, 100µl of a
1:50 cDNA sample dilution was prepared and the amount of cDNA starting material was
normalized using either Gapdh or Sdha housekeeping genes. Samples were kept at 40C for up to 5
days before they are re-normalized so the expression levels of multiple query genes could be
determined. Normally, 8µl of a 1:50 cDNA dilution was added to each well.
2.7.3 Statistical analysis
Analysis was carried out, and dissociation curves examined, using Sequence Detector System
2.1 software (Applied Biosystems, Foster City, California), as previously described. The standard
deviation for triplicate repeat wells was typically <10%. To normalize for the starting amount of
cDNA, the mean raw control gene expression was standardized by dividing the value for each
sample by the lowest mean i.e. the standardized control gene expression of the animal with the
lowest expression was defined at ‘1’. A correction value was calculated for each animal by geometric
averaging of the standardized expression values of two internal control genes (Vandesompele et al.,
2002). To normalize gene expression for each animal, the mean raw expression value of the query
gene was divided by the correction value calculated for that animal. A two-tailed Student’s t-test was
used to determine if differences in mutant and wt expression were statistically significant. Statistically
significant (p<0.05) differences in gene expression were expressed as fold-differences by dividing the
mean mutant expression by the mean wt expression. For fold-differences <1 the inverse of the
fold-difference was taken and assigned a negative value to represent down-regulation.
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2.4 Results
2.4.1 Characterization of the Rom1-/- retinal transcriptome
The retinal transcriptome of Rom1-/- was compared to Rom1+/+ mice in order to identify
differentially expressed genes that may be pathogenic, protective or bystanders to PR death. A
triplicate repeat microarray experiment was performed using pooled retinal RNA from three mice
per sample; the RNA was harvested from three month-old mutant and wild-type animals at a time-
point when 93% of PRs were still present in mutant retinas (Clarke et al., 2000a). This early time-
point was chosen because any secondary effects due to cell death in the mutant retina would be
minimal. RNA samples were then labeled and hybridized onto separate Affymetrix U74A
microarrays, scanned, and gene expression values were assigned based on hybridization intensity.
In order to compare gene expression values between Rom1-/-and wild-type retinas, raw
expression data from all six microarray experiments (three Rom1-/- and three Rom1+/+) was
normalized. The robust multi-array average (RMA) normalization method was used, which employs
background correction and quantile normalization to estimate gene expression values (Irizarry et al.,
2003). After normalization, a scatter plot was generated comparing mean log2 Rom1-/- and Rom1+/+
hybridization signals with the log2ratio of Rom1-/- to Rom1+/+ (Figure 2.1). This type of plot is also
called an M versus A plot and is a convenient way to visualize the expression values from multiple
arrays representing Rom1-/- and wild-type samples; the majority of data points are predicted to be
centered around a y-axis value of zero, indicating that the majority of genes are not differentially
expressed (Yuen et al., 2002). As expected, most data points were centered around this value,
suggesting most genes have similar expression values in both Rom1-/- and Rom1+/+ retinas. Data
points representing Rom1 had the lowest log2ratio, indicating, as expected, that it was the most
down-regulated gene in mutant retinas (Figure 2.1, arrows).
To identify significantly differentially expressed genes, significance analysis of microarrays
(SAM) was performed using the RMA normalized microarray data. The SAM algorithm incorporates
the variability of replicate arrays to calculate the likelihood that observed expression level changes
are real differences rather than artifacts (Tusher et al., 2001). The false discovery rate (FDR) is also
incorporated into the SAM algorithm to control for multiple testing. The problem of multiple
testing arises because the differentially expressed status of each gene on the array is determined by a
separate test; using the standard p<0.05 level of significance would yield
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Rom1
Rom
1-/- lo
g 2-R
om1+
/+lo
g 2
2Rom1-/-
log2+ Rom1+/+
log2
Rom1 Rom1
Rom
1-/- lo
g 2-R
om1+
/+lo
g 2R
om1-
/- log 2
-Rom
1+/+
log 2
2Rom1-/-
log2+ Rom1+/+
log2Rom1-/-
log2+ Rom1+/+
log2
Rom1
Figure 2.1: Summary of the Rom1-/- normalized microarray data M versus A scatter plot representing gene expression in three month old wild-type compared to the Rom1-/- murine model of PR degeneration. Each data point represents mean hybridization signals of Rom1-/- and wild type compared to the mean ratio of Rom1-/- to Rom1+/+ hybridization signals from three independent microarray experiments for each gene. Genes represented by data points centered around a y-axis value of zero are not differentially expressed, while data points with a mean ratio greater than or less than zero represent genes that are up-regulated or down-regulated, respectively. Arrows point to data points representing Rom1.
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approximately 5% false positives. Microarray analysis using a platform capable of detecting 20 000
genes would identify 1000 false positives amongst the differentially expressed genes if the p<0.05
level of significance were employed. The FDR reduces the number of false positives; it is calculated
after comparing the expression ratios of arrays hybridized with mutant RNA compared to wild-type
with the ratios of randomly permutated mutant and wild-type groups from the same experiment.
These permutations allow for the estimation of the number of genes that would be identified by
chance given a user-defined threshold of significant gene expression; the choice of FDR is a balance
between wanting to identify as many differentially expressed genes as possible, while minimizing the
number of predicted false positives within the group (Tusher et al., 2001).
The data-set from the microarray comparing Rom1-/- to wild-type retinas was analyzed using
SAM at a user-defined threshold such that 4.7% of the genes identified as differentially expressed
were predicted to be false positives. Twenty-nine genes were identified as significantly up-regulated
(Appendix 1), while 14 genes were identified as significantly down-regulated (Appendix 1). As
expected, Rom1 was identified as the most significantly down-regulated gene in Rom1-/- retinas
(Appendix 1). Most of the other identified genes displayed less than a two-fold difference in
expression levels. In fact, of the 29 up-regulated genes, only six were greater than 2-fold and only
one, an expressed sequence tag (EST), was greater than 3-fold up-regulated (Appendix 1). Similarly,
only one gene was down-regulated by 2-fold (Appendix 1). In addition to an estimated fold
difference, a SAM score was also assigned to each differentially expressed transcript on the basis of
the change in gene expression between microarrays hybridized with Rom1-/- compared to wild-type
retinal RNA, relative to the standard deviation of repeated measurements of random permutations
of the array data (Appendix 1). Up-regulated genes with a high score and down-regulated genes with
a low score have a greater probability of being truly differentially expressed, as opposed to being
identified by chance (Tusher et al., 2001).
To validate the microarray results, independent retinal RNA samples from Rom1-/- and
Rom1+/+ animals were reverse transcribed and used as a template in quantitative real-time polymerase
chain reactions (qPCR). All qPCR experiments were performed in triplicate; retinal cDNA samples
from three separate 3-month old Rom1-/- animals were compared to cDNA samples obtained from
three separate age-matched wild-type animals. Using independent RNA samples to validate
microarray experiments is advantageous since the biological reproducibility of each differentially
expressed transcript can be directly examined. Known genes with a high fold difference and SAM
score relative to the group were selected for qPCR analysis. Of the six genes examined, three
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transcripts were confirmed to be differentially expressed by qPCR, while two were not confirmed,
including Tmem14c, which was identified as up-regulated by SAM and down-regulated by qPCR
(Table 2.2). The lack of correlation between the microarray and qPCR results is shown on a scatter
plot comparing the log2 qPCR fold difference to log2 microarray fold-difference (Figure 2.2).
Although the slope of the trend line was positive, indicating a positive correlation between qPCR
and microarray results, the large degree of scatter suggests this microarray analysis provides only a
rough estimate of transcriptional changes that occur in Rom1-/- compared to wild-type retinas (Figure
2.2).
To quantify the degree of correlation between the qPCR and microarray results, the
concordance correlation coefficient (CCC) was employed. The CCC is an established method to test
the validity of microarray results after qPCR analysis has been performed on a subset of genes
identified as differentially expressed; a CCC of 1 indicates perfect concordance between the
microarray and qPCR fold differences, while a CCC of -1 indicates perfect reversed agreement
(Miron et al., 2006). A comparison of microarray and qPCR fold differences for the Rom1-/-
experiment yielded a CCC of 0.15 (Figure 2.2), confirming the lack of strong correlation between the
microarray and qPCR results.
In summary, the Rom1-/- transcriptome was characterized using Affymetrix oligonucleotide
microarrays, followed by RMA normalization and SAM to identify significantly differentially
expressed genes. A sample of differentially expressed genes identified by microarray analysis was
selected for confirmation by qPCR. Only three of the six selected genes were confirmed to be
differentially expressed. The 50% false positive rate is almost 10-fold higher than the predicted 4.7%
false discovery rate predicted by the SAM, resulting in a low concordance correlation coefficient of
0.15. Despite the high false positive rate, the microarray analysis suggests that the transcriptome of
the Rom1-/- retinas are minimally perturbed; 84% of genes had less than a 2-fold difference in
transcript levels between Rom1-/- and Rom1+/+ retinas (excluding Rom1 itself). The most differentially
expressed transcript was an EST (AI037577), which was up-regulated by 3.5 fold. The Rom1-/- model
of IPD displays a comparatively slow rate of PR degeneration compared to other models (Clarke et
al., 2000a). The transcriptome of a faster model of IPD, the Rds+/- mouse (Sanyal and Jansen, 1981),
was also examined to determine if a faster model of IPD displays greater differences in gene
expression when compared to Rom1-/-.
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Figure 2.2: Poor concordance between qPCR and microarray analyses in the Rom1-/- model of IPD Scatter plot comparing fold differences estimated by SAM analysis and qPCR in three-month old Rom1-/- and wild-type retinas. Each data point represents the mean log2fold-change following a triplicate repeat microarray experiment followed by SAM (x-axis) compared to the mean log2fold change following a triplicate repeat qPCR analysis (y-axis). The positive trend-line indicates a positive correlation between the microarray and qPCR analyses; the concordance correlation coefficient of 0.15 indicates that the correlation between qPCR and microarray, while positive, is poor.
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Table 2.2: Comparison of transcript fold-differences as determined by microarray and qPCR analyses in Rom1-/- and wild-type retinas.
Gene Name
Microarray fold
difference
qPCR fold
difference p value Mitochondrial ribosomal protein L16 -1.8 -1.3 0.012
Transmembrane protein 14C 2.7 -2.6 0.00021
Protein tyrosine phosphatase, receptor type, A 2.2 1.4 0.010
Transglutaminase 3, E polypeptide 2.5 3.2 4.0x10-5
Solute carrier family 17 (sodium phosphate), member 1
2.4.2 Characterization of the Rds+/- retinal transcriptome
The retinal transcriptome of the Rds+/- model of IPD was compared to Rds+/+ retinas to
identify differentially expressed genes that may be pathogenic, protective or bystanders to PR death.
A triplicate repeat microarray experiment was performed using pooled retinal RNA from 3 mice per
sample; the RNA was harvested from 7 week-old wild-type and mutant animals, a time-point when
90% of mutant PRs were still present (Clarke et al., 2000a). The isolated RNA was labeled, and each
sample was hybridized onto separate Affymetrix 430A microarrays. The arrays were then scanned,
and each gene on each array was assigned an expression value based on hybridization intensity. This
microarray experiment differs from the previously described Rom1-/- experiment in two ways: the
Rds+/- model of IPD displays faster cell death kinetics compared to Rom1-/- and the updated
Affymetrix 430A microarray platform was used compared to the U74A array.
Significantly differentially expressed genes were identified using two independent data
analysis tools. First, data analysis software provided by Affymetrix (microarray analysis suite 5 or
MAS5) was used to analyze the raw microarray data and identify differentially expressed genes. A
limitation of MAS5 is its ability to identify differences between only two individual microarrays at a
time. Since three microarrays were hybridized with Rds+/+ RNA while another three were hybridized
with Rds+/- RNA, all 9 possible pair-wise comparisons were performed using MAS5; 18 genes were
identified as differentially expressed in all comparisons (Appendix 2). The second method of data
analysis involved RMA normalization followed by SAM. An M versus A scatter plot was generated
comparing RMA normalized mean log2 Rds+/- and Rds+/+ hybridization signals with RMA normalized
log2ratio of Rds+/- to Rds+/+; data points centered around a y-axis value of zero exhibit the same level
of expression in Rds+/- and Rds+/+ retinas (Figure 2.3). While most data points were centered around
this value, several putatively differentially expressed genes were identified with log2ratios above and
below this value (Figure 2.3). The SAM algorithm was performed with the false discovery rate
adjusted to 4.7%; 128 transcripts were identified as up-regulated (Appendix 2), while 17 transcripts
were identified as down-regulated in Rds+/- retinas (Appendix 2). Significantly, all 18 genes identified
by the 9 pair-wise comparisons were also identified by SAM.
To independently validate the microarray results, RNA samples extracted from mouse
retinas not used in the microarray experiments were reverse transcribed and tested by qPCR.
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Figure 2.3: Summary of the Rds-+/- normalized microarray data M versus A scatter plot representing gene expression in 7-week old wild-type compared to the Rds+/-
murine model of PR degeneration. Each data point represents mean hybridization signals of Rds+/- and wild type compared to the mean ratio of Rds+/- to Rds+/+ hybridization signals from three independent microarray experiments for each gene. Genes represented by data points centered around a y-axis value of zero are not differentially expressed, while data points with a mean ratio greater than or less than zero represent genes that are up-regulated or down-regulated, respectively.
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Genes identified by both the 9 pair-wise comparisons and SAM were selected for further analysis. In
addition to these 18 genes, Ahrr and Socs-3 were also included in the qPCR analysis since Ahrr had a
high SAM score and Socs-3 is known to be regulated by Stat-3 (Heinrich et al., 2003), one of the 18
genes identified by the 9 pair-wise comparison and SAM. All qPCR experiments were performed
examining transcripts levels in three independent 7 week-old Rds+/- retinas compared to three
independent 7 week-old Rds+/+ retinas. A moderate degree of concordance was observed between
the qPCR and microarray results. Of the 20 examined genes, the differentially expressed status of 12
transcripts was confirmed by qPCR (Table 2.3). To directly compare the microarray and qPCR
results, a scatter plot was generated comparing the log2 qPCR fold differences to the log2 microarray
fold differences (Figure 2.4). The positive trend line indicates that the microarray and qPCR data are
positively correlated and CCC analysis revealed a value of 0.49, although this value may be slightly
inflated due to a single data point (Figure 2.4, arrow), representing a highly differentially expressed
gene identified by both microarray and qPCR. Microarray analysis tends to underestimate the actual
fold difference as determined by qPCR (Yuen et al., 2002; Chowers et al., 2003). Consistent with this
tendency, the fold differences determined by qPCR were higher than the predicted microarray fold
difference in 11 out of 12 examples.
Due to the large number of significantly differentially expressed genes, the Ingenuity
pathway analysis software was used to identify any relationships between the identified genes. The
Ingenuity pathway finding algorithm functions by searching for relationships within a user-defined
set of molecules- in this case, the differentially expressed genes identified by SAM- using a database
of known interactors. Networks that maximize the interconnectedness of the user-defined molecules
of interest with all the molecules in the ingenuity database are then created. Additional molecules
from the Ingenuity database are used to specifically connect two or more smaller networks by
merging them into a larger one. The 145 significantly differentially expressed genes identified by
SAM were analyzed by the Ingenuity pathway analysis algorithm and 6 networks were identified
among the differentially expressed genes that function in a variety of different processes, including:
cell and immune signalling (Appendix 2.5); molecular transport, cancer and cell death (Appendix
2.6); immune and injury response (Appendix 2.7); intercellular signalling and interaction (Appendix
2.8); immune and inflammatory disease (Appendix 2.9), and cellular growth proliferation (Appendix
2.10).
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Table 2.3: Comparison of transcript fold-differences as determined by microarray and qPCR analyses in Rds+/- and wild-type retinas. Genes represented on the microarray with multiple probe sets were assigned multiple fold differences.
Gene symbol Microarray
Fold Difference
qPCR Fold Difference
p-value (qPCR)
Endothelin-2 8.34 32.00 0.00056 Oncostatin M receptor 2.30
1.25 2.30 3.0x10-5
Solute carrier family 29 (nucleoside transporters), member 1
Suppressor of cytokine signalling 3 1.56 1.17 1.26
2.80 2.80
0.001
Aryl hydrocarbon receptor 2.30 1.36 0.025
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Figure 2.4: Moderate concordance between qPCR and microarray analyses in the Rds+/- model of IPD. Scatter plot comparing fold differences estimated by SAM analysis and qPCR in 7-week old Rds+/- and wild-type retinas. Each data point represents the mean log2fold-change following a triplicate repeat microarray experiment followed by SAM (x-axis) compared to the mean log2fold change following a triplicate repeat qPCR analysis (y-axis). The positive trend-line indicates a positive correlation between the microarray and qPCR analyses; the concordance correlation coefficient of 0.49 indicates that the correlation between qPCR and microarray is quite good. Arrow indicates Endothelin-2, the most highly differentially expressed gene by microarray and qPCR analyses.
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The highly significant (p<10-50) up-regulated network involved in cell and immune signalling
(a putative IL-6 pathway) was the most statistically significant and was comprised of transcripts with
some of the highest estimated fold differences, suggesting this network is playing an important role
in Rds+/- retinas (Appendix 2.5). One highly differentially expressed gene not included in this
network is endothelin-2 (32-fold up-regulated), which was identified in a network involved in cellular
growth and proliferation (Appendix 2.10).
In conclusion, the Rds transcriptome was characterized using Affymetrix oligonucleotide
microarrays, followed by two different data analysis strategies. Of the 20 genes selected for qPCR
validation, 12 were confirmed to be significantly up-regulated in Rds+/- retinas. This false positive rate
of 40% (8/20) is significantly greater than the 4.7% false discovery rate predicted by the SAM. The
Ingenuity pathway finding algorithm was then employed to determine if any of the 145 differentially
expressed genes identified by SAM participated in any common networks. Of the 6 putative
pathways identified, a pathway involved in immune and cell signalling was identified that
encompassed a large number of significantly differentially expressed transcripts at comparatively
high fold differences, suggesting it is playing an important role in Rds+/- retinas.
2.5 Discussion
Significantly differentially expressed transcripts were identified in both the Rom1-/- and Rds+/-
retinas. Remarkably, none of the identified genes were differentially expressed in both models. This
lack of overlap was surprising because Rom1 and Rds are homologs and both encode proteins that
associate to form oligomers thought to play a structural role in the outer segment of the retina
(Connell et al., 1991; Bascom et al., 1992; Clarke et al., 2000b). The Rds+/- model of IPD displays
much faster cell death kinetics compared to the Rom1-/- model, which exhibits one of the slowest
rates of degeneration of any IPD model (Clarke et al., 2001). One possible interpretation of the lack
of overlapping differentially expressed genes is that the rate of degeneration may be an important
factor in determining the magnitude, number and type of transcriptional changes occurring in IPD
retinas; the functional category of the protein encoded by the IPD gene may be less important in
determining the transcriptional response. The observation that several common transcriptional
responses have been observed in different models, all displaying cell death kinetics faster than the
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Rom1-/- model, supports this hypothesis. For example, members of the IL-6 pathway were up-
regulated in response to light damage, mechanical stress, and the Rd1-/- model of IPD (Chen et al.,
2004; Vazquez-Chona et al., 2004; Rattner and Nathans, 2005; Zacks et al., 2006).
Differentially expressed genes identified in Rds+/- retinas by microarray analysis were more
likely to be biologically reproducible when compared to the genes identified in Rom1-/- retinas; the
Rom1-/- microarray-qPCR comparison yielded more false positives and a lower CCC compared to the
Rds+/- microarray-qPCR comparison. The different microarray platforms used for each experiment
could be one source of the differing reproducibility. The Rom1-/- microarray experiment employed
Affymetrix U74A arrays, which contained probes designed to interrogate the ~12 000 genes and
ESTs present in the unigene database build 71 (March, 2000)(Affymetrix, 2004). In comparison, the
Rds+/- microarray experiment was performed using the more advanced 430A array, containing
probes representing ~14 000 genes and ESTs, many of which were redesigned based on the draft
sequence of the mouse genome and the updated sequences contained within the unigene database
build 107 (June, 2002)(Affymetrix, 2004). The microarray platform itself may have also been
improved between the U74A and 430A, although no specific details were released by the company.
Thus, the use of the 430A array may have facilitated the identification of more validated
differentially expressed genes in Rds+/- retinas.
Both the MAS5 9 pair-wise comparison and SAM identified an overlapping set of 18
differentially expressed genes in the Rds+/- model of IPD. This overlap is significant because each
method of data analysis is quite different. For example, MAS5 employs a normalization technique
based on scaling the hybridization intensities from each microarray being compared, so that the
average is the same (Affymetrix, 2004). In contrast, RMA involves a quantile normalization method
such that only genes with similar hybridization intensities are used in the normalization process
(Irizarry et al., 2003). The MAS5 algorithm computes the hybridization intensity for each gene by
identifying the degree to which RNA binds to its perfect match probe compared to the mismatch
probe- each perfect match probe has a mismatch negative control- on the microarray (Affymetrix,
2004). In contrast, the SAM algorithm ignores the mismatch data and identifies differentially
expressed genes based on the perfect match hybridization intensities only. The fact that two such
different data analysis tools were able to identify an overlapping set of differentially expressed genes,
suggests that the identified genes are likely to be truly differentially expressed. The fact that only
10/18 genes identified by both methods were verified by qPCR suggests an unknown bias in either
59
the microarray platform itself, or a difference in the RNA samples used for the microarray analysis
compared to qPCR.
The genetic background of the Rom1-/- and Rds+/- animals may have also contributed to the
variability of the microarray results. While Rds+/- and Rds+/+ mice are inbred animals (Schalken et al.,
1990; jackson-laboratories, 2008), Rom1-/- and its wild-type control are outbred (CD1 and 129 mix)
(Clarke et al., 2000b). The Rom1-/-microarray experiments were performed using pooled samples,
which is known to decrease variability by mitigating the possible effect of a single outlier (Allison et
al., 2006). However, genetic modifiers are known to affect the rate of cell death in many models of
IPD (Pacione et al., 2003); therefore, the lack of genetic homogeneity within and between Rom1-/-
and Rom1+/+ animals may have increased the variability in the Rom1-/- microarray experiment.
Increased variability would manifest itself as a decrease in the number of identified significantly
differentially expressed genes; increased variability would make it less likely that a given transcript
would be identified as significantly differentially expressed. The Rds+/- microarray analysis yielded
more reproducible results, a greater number of significantly differentially expressed genes with
higher fold differences. Additionally, six networks were identified among the differentially expressed
genes identified in Rds+/- retinas; no pathways were identified among the differentially expressed
genes identified in the Rom1-/- model. As such we decided to focus on the results obtained from the
Rds+/- model.
Six networks were identified as up-regulated in Rds+/- retinas (Appendix 2.5), including: 1) a
network involved in cell and immune signalling (a putative IL-6 pathway), featuring signal transducer
and activator of transcription (STAT) signalling, which is known to be activated when IPD retinas
are treated with neurotrophic factors that slow the rate of cell death (Chaum, 2003); 2) a network
involved in cancer and cell death including the proto-oncogene B-cell CLL/lymphoma 3 (BCL3)
and transforming growth factor beta (Tgf-b), which is known to mediate cell death in the retina are
members of this pathway (Duenker, 2005). Neuroprotective proteins were also identified in this
pathway, including GADD45, a DNA-damage inducible gene thought to play a protective role in
damaged neurons and platelet derived growth factor (Pgdf), known the protective PI 3-kinase/Akt
pathway in the retina were both (Torp et al., 1998; Biswas et al., 2008). Thus, this pathway consists
of both neuroprotective and pathogenic components; 3) a pathway involved in viral function,
immune response and injury was also up-regulated. Tumor necrosis factor is a major node in this
pathway and is a known pro-apoptotic factor involved in systemic inflammation and known to be
involved in neuronal cell death (Venters et al., 2000); 4) a cell signalling pathway featuring beta-
60
estradiol, which has recently been shown to exhibit neuroprotective properties on retinal ganglion
cells (Zhou et al., 2007); 5) an additional inflammatory pathway featuring STAT and mitogen
activated protein kinase signalling, generally thought be protective in the retina (Wahlin et al., 2001;
Chaum, 2003); 6) finally, the pathway involved in cellular growth and differentiation includes tumour
protein 53 (p53), which is involved in regulating the cell cycle, especially during DNA damage. It has
been suggested that p53 controls the activity of C/EBPδ, a transcription factor involved in
apoptosis during mammary gland development (Thangaraju et al., 2005).
The putative IL-6 pathway involved in cell and immune signalling (pathway #1 above) was
the most significant network identified. The pathway included more genes compared to the other
five pathways; 24 of the 145 significantly differentially expressed transcripts identified by the
microarray analysis were present in this pathway. As such, it was the most statistically significant of
all the pathways (p<10-50), since the likelihood that 24 genes that make up a common pathway could
randomly be present in a group of 145 genes is very low.
IL-6 pathway activation (Figure 2.5) may represent a near universal response to cell stress
and is probably involved in PR survival (Ueki et al., 2008). Our report represents one of the few
studies that have recognized increased IL-6 signalling in IPD retinas. For example, Rattner et al.
listed the Oncostatin M receptor (Osmr), a member of the IL-6 family of receptors, Stat-3, and
C/Ebpδ, a gene known to be regulated by Stat-3, as up-regulated in prCAD-/-retinas; however, they
were never identified as being a part of the same pathway (Rattner and Nathans, 2005). In contrast, a
recent report did identify increased IL-6 pathway activation in IPD retinas; cardiotrophin-like
cytokine (clc), and leukemia inhibitory factor (lif), both IL-6 cytokines, were up-activated
concomitant with increased phosphorylated Stat-3, an indicator of IL-6 signalling (Samardzija et al.,
2006). IL-6 pathway activation has also been reported in response to mechanical stress (Vazquez-
Chona et al., 2004; Zacks et al., 2006) and light mediated damage (Chen et al., 2004; Rattner and
Nathans, 2005). Several studies have shown that treatment with IL-6 cytokines, including ciliary
neurotrophic factor and cardiotrophin-1, have resulted in Stat-3 phosphorylation and increased PR
survival, suggesting endogenous IL-6 signalling may be protective in the retina (LaVail et al., 1998;
Song et al., 2003).
We selected the putative IL-6 pathway for additional analysis for several reasons. First, the
pathway the most significant compared to the other pathways identified by Ingenuity analysis.
Secondly, there is evidence in the literature to suggest that the IL-6 pathway is protective; its up-
regulation in a variety of contexts also makes IL-6 signalling an important therapeutic target. Finally,
61
loss-of-function mouse mutants were available for several genes in the IL-6 pathway, facilitating
genetic experiments to test the role of pathway members in IPD. The subject of the next chapter is
the use of genetic analysis to determine whether the up-regulation of several IL-6 pathway members
is mechanistically linked to cell survival.
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Figure 2.5: Up-regulation of a putative IL-6 cytokine pathway in Rds+/- retinas Lif and Osm bind to Lifr and Osmr, respectively, which both share gp130 as a common co-receptor. Upon ligand binding, tyrosine kinases (Jak1,Jak2 and Tyk2) associated with gp130, Osmr and Lifr become activated. Once active, these kinases can phosphorylate Stat-3, which then dimerizes and is transported to the nucleus, where it acts as a transcriptional activator of several genes, including: Glial fibrillary acidic protein (Gfap), α-2 macroglobulin (α-2m), Suppressor of cytokine signalling (Socs3), CAAAT enhancer binding protein δ (C/EBPδ). Socs-3 is a negative regulator of Stat-3 phosphorylation. All fold differences are qPCR values.
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Appendix 2.1: Up-regulated transcripts in the retinas of Rom1-/- mice
Accession Number
Gene Name SAM Score
SAM Numerator
Fold Change
96222_at AI037577 7.97 1.83 3.54 96353_at Transmembrane protein 14C
(Tmem14c) 6.86 1.44 2.73
100908_at Protein tyrosine phosphatase, receptor type A (Ptpra)
Appendix 2.5: Up-regulated network involved in cell and immune signalling
Twenty-four differentially expressed genes identified from the microarray analysis are included in this network (p<10-50). Red shading indicated an up-regulated gene as identified by microarray analysis while un-shaded circles are genes included by the ingenuity software package with a known relationship with one of the differentially expressed genes. Solid lines indicate a direct relationship such as a protein-protein interaction while dashed lines indicates an indirect relationship. Abbreviations: aryl hydrocarbon receptor (AHR), aryl-hydrocarbon receptor repressor (AHRR), ATPase, Na+/K+ transporting, alpha 1 polypeptide (ATP1A1), betacellulin (BTC), CCAAT/enhancer binding protein (C/EBP), delta (CEBPD), colony stimulating factor 1 receptor (CSF1R), cathepsin S (CTSS), cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1), dopachrome tautomerase (DCT), FXYD domain containing ion transport regulator 1 (FXYD1), glial fibrillary acidic protein (GFAP), interferon induced transmembrane protein 2,3,9 (IFITM2,3,9), janus kinase (JAK), jun B proto-oncogene (JUNB), MAP kinase interacting serine/threonine kinase 1 (MKNK1), oncostatin M receptor (OSMR), S100 calcium binding protein A1 (S100A1), serine (or cysteine) peptidase inhibitor, clade A, member 3N (SERPINA3N), suppressor of cytokine signalling 3 (SOCS3), signal transducer and activator of transcription 1 (STAT1), TNFAIP3 interacting protein 2 (TNIP2), tyrosinase-related protein 1 (TYRP1).
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Appendix 2.6: Up-regulated network involved in molecular transport, cancer and cell death
Twenty-two differentially expressed genes identified from the microarray analysis are included in this network (p<10-44). Red shading indicated an up-regulated gene as identified by microarray analysis while un-shaded circles are genes included by the ingenuity software package with a known relationship with one of the differentially expressed genes. Solid lines indicate a direct relationship such as a protein-protein interaction while dashed lines indicates an indirect relationship. Abbreviations: alpha-2-macroglobulin (A2M), ADAM metallopeptidase with thrombospondin type 1 motif, 1 (ADAMTS1), activating transcription factor 3 (ATF3), beta-2-microglobulin (B2M), B-cell CLL/lymphoma 3 (BCL3), complement component 1, q subcomponent, A,B,C chain (C1QA,B,C), ceruloplasmin (ferroxidase) (CP), growth arrest and DNA-damage-inducible, beta (GADD45B), guanylate binding protein 2, interferon-inducible (GBP2), histocompatibility 2, D region (H2-LD), major histocompatibility complex, class I, C,E (HLA-C,E), metallothionein 1E (MT1E,F), procollagen C-endopeptidase enhancer (PCOLCE), platlet derived growth factor (Pdgf), podoplanin (PDPN), POU class 2 homeobox 2 (POU2F2), serpin peptidase inhibitor, clade G (C1 inhibitor), member 1 (SERPING1), solute carrier family 29 (SLC29A1), transforming growth factor (Tgf), thrombospondin 1 (THBS1)
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Appendix 2.7: Up-regulated network involved in viral function, immune response and injury
Sixteen significantly differentially expressed genes identified from the microarray analysis are included in this network (p<10-29). Red shading indicated an up-regulated gene as identified by microarray analysis while un-shaded circles are genes included by the ingenuity software package with a known relationship with one of the differentially expressed genes. Solid lines indicate a direct relationship such as a protein-protein interaction while dashed lines indicates an indirect relationship. Abbreviations: ATP-binding cassette, sub-family C member 3 (ABCC3), arachidonate 5-lipoxygenase-activating protein (ALOX5AP), asparagine synthetase (ASNS), BUB1,3 budding uninhibited by benzimidazoles 1 homolog beta (yeast) (BUB1,3), chemokine (C-C motif) receptor 2 (CCR2), CCAAT/enhancer binding protein (C/EBP), beta (CEBPB), carboxypeptidase B2 (plasma) (CPB2), deoxynucleotidyltransferase, terminal, interacting protein 2 (DNTTIP2), estrogen receptor 1 (ESR1), Fc fragment of IgG, receptor, transporter, alpha (FCGRT), guanylate nucleotide binding protein 1 (GBP1), guanylate binding protein 2, interferon-inducible (GBP2), guanylate binding protein 4 (GBP4), guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 2 (GNAI2), guanine nucleotide binding protein (G protein), beta polypeptide 3 (GNB3), G-protein signalling modulator 3 (AGS3-like, C. elegans) (GPSM3), hepcidin antimicrobial peptide (HAMP), haptoglobin-related protein (HPR), interferon induced transmembrane protein 3 (1-8U) (IFITM3), interferon, beta 1, fibroblast (IFNB1), interleukin 3 receptor, alpha (low affinity) (IL3RA), ladinin 1 (LAD1), mesoderm specific transcript homolog (mouse) (MEST), macrophage activation 2 like (MPA2L), msh homeobox 2 (MSX2).
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Appendix 2.8: Up-regulated network involved in cell-cell signalling
Fifteen significantly differentially expressed genes identified from the microarray analysis are included in this network (p<10-27). Red shading indicated an up-regulated gene as identified by microarray analysis while un-shaded circles are genes included by the ingenuity software package with a known relationship with one of the differentially expressed genes. Solid lines indicate a direct relationship such as a protein-protein interaction while dashed lines indicates an indirect relationship. Abbreviations: amiloride-sensitive cation channel 3 (ACCN3), ATP/GTP binding protein 1 (AGTPBP1), aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2), bone marrow stromal cell antigen 1 (BST1), chromosome 10 open reading frame 54 (C10ORF54), CD46 molecule, complement regulatory protein (CD46), cytokine receptor-like factor 2 (CRLF2), discs, large homolog 4 (DLG4), enolase 1, (alpha) (ENO1), Fc fragment of IgG, receptor, transporter, alpha (FCGRT), forkhead box P3 (FOXP3), guanylate binding protein 2, interferon-inducible (GBP2), glial fibrillary acidic protein (GFAP), histocompatibility 2, Q region locus 5,8 (H2-Q5,8), major histocompatibility complex, class I, C (HLA-C), interleukin 6 (IL6), lin-7 homolog B (C. elegans) (LIN7B), lymphocyte antigen 86 (LY86), microRNA 21 (MIRN21 (includes EG:406991)), nuclear receptor co-repressor 2 (NCOR2), 3-oxoacid CoA transferase 1 (OXCT1), pre-B-cell leukemia homeobox interacting protein 1 (PBXIP1), platelet-derived growth factor alpha polypeptide (PDGFA), PDZ and LIM domain 4 (PDLIM4), PDZ domain containing 1 (PDZK1), polypyrimidine tract binding protein 2 (PTBP2), protein tyrosine phosphatase, non-receptor type 13 (APO-1/CD95 (Fas)-associated phosphatase) (PTPN13), RD RNA binding protein (RDBP).
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Appendix 2.9: Up-regulated network involved immunological and inflammatory disease
Twelve significantly differentially expressed genes identified from the microarray analysis are included in this network (p<10-20). Red shading indicated an up-regulated gene as identified by microarray analysis while un-shaded circles are genes included by the ingenuity software package with a known relationship with one of the differentially expressed genes. Solid lines indicate a direct relationship such as a protein-protein interaction while dashed lines indicates an indirect relationship. Abbreviations: anthrax toxin receptor 2 (ANTXR2), complement component 4B (C4B), Casein kinase 2, Casein Kinase II, CKII (Ck2), clusterin (CLU), collagen, type VIII, alpha 1 (COL8A1), cAMP response element binding (Creb), DnaJ (Hsp40) homolog, subfamily C, member 11 (DNAJC11), elongation protein 2 homolog (S. cerevisiae) (ELP2), heat shock protein 90 (Hsp90), interleukin 31 (IL31), interleukin 9 receptor (IL9R), keratin 72 (KRT72), low density lipoprotein (LDL), MAP kinase protein (Mapk), oncostatin M receptor (OSMR), platelet-activating factor acetylhydrolase, isoform Ib, alpha subunit (PAFAH1B1), PI3 kinase (PI3K), protein kinase C (Pkc), protein tyrosine phosphatase type IVA, member 3 (PTP4A3), retinol binding protein 4, plasma (RBP4), suppressor of cytokine signalling 4 (SOCS4), signal transducer and activator of transcription 3 (STAT3), transcription elongation factor B3 (TCEB3), transferrin (TF), transthyretin (TTR), ubiquitin-like, containing PHD and RING finger domains, 1 (UHRF1).
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Appendix 2.10: Up-regulated network involved in cellular growth and proliferation
Twelve significantly differentially expressed genes identified from the microarray analysis are included in this network (p<10-27). Red shading indicated an up-regulated gene as identified by microarray analysis while un-shaded circles are genes included by the ingenuity software package with a known relationship with one of the differentially expressed genes. Solid lines indicate a direct relationship such as a protein-protein interaction while dashed lines indicates an indirect relationship. Abbreviations: aurora kinase B (AURKB), Bruton agammaglobulinemia tyrosine kinase (BTK), cyclin-dependent kinase inhibitor 1B (p27, Kip1) (CDKN1B), CCAAT/enhancer binding protein (C/EBP), delta (CEBPD), crystallin, mu (CRYM), endothelin 2 (EDN2), endothelin receptor type A (EDNRA), etoposide induced 2.4 mRNA (EI24), fibulin 5 (FBLN5), growth arrest-specific 1 (GAS1), glucuronidase, beta (GUSB), hexokinase 2 (HK2), interleukin 31 (IL31), integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12) (ITGB1), mannosidase, alpha, class 2B, member 1 (MAN2B1), mitogen-activated protein kinase 1 (MAPK1), melanoma inhibitory activity (MIA), myeloid leukemia factor 1 (MLF1), v-myc myelocytomatosis viral oncogene homolog (MYC), myosin X (MYO10), phosphomevalonate kinase (PMVK), protein phosphatase 1, regulatory (inhibitor) subunit 15A (PPP1R15A), prostaglandin D2 synthase (PTGDS), prostaglandin F receptor (PTGFR), poliovirus receptor-related (3PVRL3), regulatory factor X, 2,3 (RFX2,3), ribonuclease, RNase A family, 4 (RNASE4).
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3 A role for IL-6 signalling molecules in inherited photoreceptor degenerations
The majority of the work presented in this chapter is my own, with the following exceptions:
1) Figure 3.2 and Figure 3.7c: Immunoblots were performed under my supervision by Lynda Ploder
2) Figure 3.5: laser capture microdissection and qPCR was performed by myself, Coco Jiang and
Alexa Bramall.
3) Figure 3.7b: Alexa Bramall performed the sectioning and staining of retinas from the Lif+/+ and
Lif-/- mice.
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3.1 Abstract
We previously reported that in inherited photoreceptor degenerations (IPDs), the mutant
photoreceptors (PRs) are at a constant risk of death (Clarke et al. Nature 2000). Using microarrays
and quantitative PCR (qPCR) to identify genes that mediate the constant risk, we found up-
regulation of a putative IL-6 cytokine pathway in 3 mutant PR mouse models, when 60-90% of PRs
are still alive. For example, in the Rds+/- model, Oncostatin M (2X up by qPCR) → Oncostatin M receptor
(Osmr)(2.6X up) → Stat3 (2.3X up) → the transcription factor C/EBPδ (3.2X up), with increases in
the cognate proteins Osmr (3X up), Stat3 (2.6X up), and the phosphorylated, transcriptionally
active form of Stat-3 (5.8X up)(all p<0.01). Leukemia inhibitory factor (Lif) mRNA, another IL-6
cytokine was also found to be up-regulated (3.0X up). These increases occurred predominantly in
Müller glia, but the increase in C/EBPδ mRNA was in several cell types, including PRs. Since
exogenous IL-6 cytokines have been shown to slow PR death and also increase Müller cell pStat-3,
we asked whether the endogenous increases in IL-6 pathway genes in mutant retinas were a survival
response, and generated mutant PR models with Osmr, Lif or C/EBPδ loss-of-function (LOF)
mutations. Osmr LOF decreased PR survival in the retinas of: 4 month old Rds+/-;Osmr-/- mice had
12.5% fewer PRs than those of Rds+/-;Osmr+/+ mice (n=9, p<0.05), and the retinas of 31 day-old Tg-
RHO(P347S);Osmr-/- mice had 13.5% fewer PRs (n=6, p<0.01). The putative IL-6 response pathway
(above), if predominantly activated through the Osmr, would be expected to exhibit a decline in
pStat3 levels in the absence of Osmr. Unexpectedly, however, Osmr LOF had no effect on pStat3
levels in Rds+/-;Osmr-/- retinas, indicating that Stat3 activation is mediated predominantly through IL-
6 cytokines other than Osm, or other pathways. In contrast to the Osmr LOF, Lif LOF increased
mutant PR survival in the retinas of: PN13 Rd1-/-;Lif -/- mice had 14% more PRs than Rd1-/-;Lif+/+
mice (n=6, p<0.003) and a 1.7 fold decrease in pStat-3 (n=4, p<0.05). Similarly, C/EBPδ LOF
increased mutant PR survival in the retinas of: 8-month old Rds+/-;C/EBPδ-/- mice had 18% more
PRs than Rds+/-;C/EBPδ+/+ mice (n=5, p<0.005). These findings suggest that in mutant PRs 1) up-
regulation of the Osmr receptor is a survival response; 2) the presence of Lif or C/EBPδ is
pathogenic, and therefore 3) Osmr , Lif and C/EBPδ act either in different pathways or different
cells to account for the differing effects of their LOF on PR cell death; 4) the partial effects of Osmr,
Lif and C/EBPδ LOF indicate that other genes also mediate the constant risk of death.
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3.2 Introduction
Photoreceptors are highly specialized cells in the retina capable of biochemically responding
to visual cues, beginning a cascade of events required for vision (Burns and Arshavsky, 2005;
Fernald, 2006). They are also particularly susceptible to genetic insults. For example, more than 193
different loci, including 142 cloned genes, have been implicated in IPD, a major cause of visual
impairment for which no therapy has been effective, except for the recent gene therapy outcomes
for Leber’s congenital amaurosis (Acland et al., 2001; Dejneka et al., 2004; Hartong et al., 2006;
Daiger et al., 2008; Hauswirth et al., 2008). One promising approach is treatment with neurotrophic
factors, since their exogenous administration promotes PR survival in a variety of animal models
(Faktorovich et al., 1990; LaVail et al., 1992; LaVail et al., 1998).
Photoreceptors are partially protected by treatment with neurotrophic factors in animal
models of PR degeneration (LaVail et al., 1998; Chaum, 2003). PR protection was first demonstrated
in the RCS rat model of IPD, in which a single intravitreal injection of basic fibroblast growth factor
(bFGF) slowed the rate of PR degeneration; two months after treatment, five to seven rows of PR
nuclei remained, compared to one row of nuclei remaining in untreated RCS controls (Faktorovich
et al., 1990). Exposing rodents to bright light also induces PR degeneration. In a light-induced model
of PR degeneration, bFGF also had a significant protective effect (Faktorovich et al., 1992; LaVail et
al., 1992; O'Driscoll et al., 2008). LaVail et al. then used the light-induced model of IPD to
determine if other neurotrophic factors could also provide PR protection; of the 8 factors tested,
four provided a high degree of PR rescue, including interleukin 1β (IL-1β), acidic fibroblast growth
fold) and C/EBPδ (3.2-fold) were demonstrated to be up-regulated (Table 3.2). The transcript level
of Osm, the ligand specific to the Osmr, was then examined by qPCR analysis since probes for this
gene were not present on the microarrays employed; Osm mRNA was 2-fold increased in Rds+/-
retinas (Table 3.2). To ascertain whether the up-regulation of Osm and the Osmr was a specific
response among IL-6 cytokines, I examined the levels of the other IL-6 cytokines and receptors
were examined in Rds+/- compared to wild-type retinas. Of the six receptors and 8 cytokines in the
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Table 3.2: Quantification of selected differentially expressed transcripts in three different mouse models of IPD
All fold-differences were calculated based on the average difference of a triplicate repeat microarray or qPCR experiment; p values were determined using a two-tailed t test. The microarray experiment yielded multiple fold differences for Gfap and Stat-3 due to the presence of additional probes on the microarray. Abbreviations: Oncostatin M (Osm), Leukemia inhibitory factor (Lif), Oncostatin M receptor (Osmr), Leukemia inhibitory factor receptor (Lifr), Glycoprotein 130 (gp130), Signal transducer and activators of transcription-3 (Stat-3), Suppressor of cytokine signaling-3 (Socs-3), Alpha-2-macroglobulin (a2M), CCAAT enhancer binding protein delta (C/EBPδ), Glial fibrillary acidic protein (Gfap).
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IL-6 family, I found Lif to be 3-fold up-regulated, while gp130 and the IL-6 receptor were up-
regulated 1.3-fold and 1.4-fold, respectively, in Rds+/- retinas (Figure 3.1). Interestingly, the
microarray analysis failed to identify increased Osm, Lif, gp130 or IL-6r, suggesting that the
microarray analysis did not identify all differentially expressed genes. Thus, these results show that a
specific subset of IL-6 cytokines and receptors are up-regulated in Rds+/- retinas. Of particular
interest are Osm, the Osmr, and Lif since they are differentially expressed to the greatest degree. The
oncostatin ligand and its receptor, Osmr, also represent the only up-regulated cytokine-receptor pair.
To establish whether the up-regulation of Lif, Osm, the Osmr and their downstream putative
pathway members are a general response to mutations leading to IPDs, I then interrogated two
additional well-characterized mouse models of IPD, to determine if the same IL-6 cytokine
responses were occurring. The two models include a mouse line expressing a human mutant
rhodopsin trangene with a proline to serine substitution at amino acid 347 (P347S) (Li et al., 1996),
and the Rd1 model, which has a defect in β-phosphodiesterase (Chang et al., 2002). RNA was
harvested from both models at an early time-point when ~60% of PRs were present in the P347S
model while >90% of PRs were present in the Rd1 model. Early time-points were studied to
minimize the secondary effects of apoptosis. All the same IL-6 cytokine-related molecules up-
regulated in the Rds+/- retina were found to be differentially expressed in both P347S and Rd1-/-
retinas (Table 3.2), with two notable exceptions: 1) Osm was up-regulated in the Rds+/- and P347S
models but not the Rd1 -/- model, and 2) the Lifr, which was not differentially expressed in the Rds+/-
or P347S models, was down-regulated in the Rd1-/- model by 1.6-fold (Table 3.2). Interestingly, there
was a positive correlation between the rate of degeneration of the IPD model and the magnitude of
fold change for the examined pathway members. For example, the Osmr was 2.6-fold up-regulated in
the comparatively slow Rds+/- model, while it was up-regulated 3.4-fold and 5.6-fold in P347S and
Rd1-/- retinas, respectively; P347S retinas exhibit an intermediate rate of cell death between Rds+/- and
Rd1-/- retinas. In summary, many components of the IL-6 putative pathway were up-regulated in all
three models of IPD, suggesting that their up-regulation is a general response to the presence of a
PR mutation. Furthermore, the degree to which the pathway members were up-regulated correlated
with the rate of degeneration.
To determine if the changes in the expression of IL-6 cytokines related mRNAs led to
elevations of the cognate proteins in Rds+/- retinas, I performed immunoblot analyses. The Osmr
and Stat-3 exhibited a 3.1-fold up-regulation and 2.6-fold up-regulation in Rds+/- retinas,
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Figure 3.1: Several IL-6 cytokines and receptors are up-regulated in the retinas of Rds+/- mice Quantitative PCR indicates that Oncostatin M (Osm), Oncostatin M receptor (Osmr), Glycoprotein 130 (Gp130), leukemia inhibitory factor (LIF), IL-6 receptor (IL6r) are up-regulated in Rds+/- retinas; the other IL-6 cytokines and receptors were either not detectable by qPCR or were not differentially expressed. All fold-differences were calculated based on the average difference of a triplicate repeat microarray or qPCR experiment; p values were determined using a two-tailed t test. The Lif receptor (Lifr) can bind Cardiotrophin-1 (Ct-1), Cardiotrophin-like cytokine (Clc), and Lif, while Ciliary neurtrophic factor receptor (Cntfr) can bind Cntf and neuropoietin (Np). All other receptors can bind only a single ligand.
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Figure 3.2: Rds+/- retinas exhibit increased IL-6/Jak-Stat signaling Retinal proteins from wild-type (lanes 1-3) and Rds+/- mice (lanes 4-6) were extracted from 7 week old mice. Homogenized retinas were subject to immunoblot assays to detect members of the Jak-Stat signaling pathway. Densitometry analysis revealed: a 3.1 fold up-regulation of Osmr in Rds+/- (n=3, 25048 pixels+/- 3439) compared to Rds+/+ retinas (n=3, 8021 pixels +/- 712; p<0.01); ERK-1,2 and pERK-1,2 levels were unchanged in Rds+/- retinas. Stat-1 was 1.9-fold up-regulated in Rds+/- (n=3, 34941.2 pixels+/-6186) compared to Rds+/+ retinas (n=3, 18427.4 pixels+/-8917; p<0.02). A 5.85-fold up-regulation of pStat-3 in Rds+/- (n=3, 17518 pixels +/- 1870) compared to Rds+/+ (n=3, 2993 pixels +/- 639; p<0.002); and Stat-3 displays a 2.6 fold up-regulation in Rds+/- retinas (n=3, 15464 pixels +/- 1730) compared to Rds+/+ retinas (n=3, 5852 pixels +/- 1482; p<0.015). All pixels were followed by +/- standard error of the mean and all p values were calculated using a two-tailed t test.
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respectively (Fig. 3.2), concordant with the qPCR results. Since the activity of the putative pathway
depends on the phosphorylation state of Stat-3 (Stat-3 must be phosphorylated to function as a
transcription factor (Heinrich et al., 1998; Levy and Lee, 2002)), I also examined the levels of
phosphorylated Stat-3 (pStat-3) in wild-type and Rds+/- retinas. I found a 5.85-fold up-regulation of
pStat-3 in Rds+/- retinas (Fig. 3.2), an increase distinctly higher then the 2.6-fold increase observed
for Stat-3. Interestingly, pStat-1, which is capable of forming heterodimers with pStat-3 (Heinrich et
al., 1998), was below the level of detection in Rds+/- and wild-type retinas, although Stat-1 was up-
regulated by 1.9-fold in the retinas of Rds+/- mice. In conclusion, these results show that protein
levels of key polypeptides of the putative pathway are up-regulated in Rds+/- retinas, and that the
putative pathway that includes pStat-3 is more active in Rds+/- retinas.
The IL-6 family of cytokines are also capable of directly activating the extracellular signal-
regulated kinases 1,2 (ERK-1,2) (Kamimura et al., 2003; Song et al., 2003). To determine if ERK-1,2
are up-regulated in Rds+/- retinas, I again used immunoblot analysis. No difference in total ERK-1,2
abundance or in that of the active phosphorylated form of ERK-1,2 (pERK-1,2) was identified,
suggesting that ERK-1,2 are not activated in this context (Fig. 3.2).
Müller glia have been shown to express Stat-3 and pStat-3 in response to exogenous cytokine
treatment (Peterson et al., 2000; Song et al., 2003; Ueki et al., 2008). To ascertain the cell type(s) in
which up-regulated Osmr, Stat-3 and pStat-3 occurs in IPD, I first performed immunostaining of
Rds+/- retinas. The Osmr was up-regulated in both astrocytes and Müller glia of Rds+/- retinas (Fig 3.3
A,B), as determined by co-localization studies with glial fibrillary acidic protein (Gfap), a marker of
Müller glia projections and astrocytes (Figure 3.3 C,D) (Chen and Weber, 2002). In contrast, to wild-
type and Rds+/- retinas, no Osmr staining was observed in the Osmr-/- retinas, confirming the
specificity of this antibody (Fig 3.3 M). While Stat-3 staining in wild-type retinas was below the level
of detection (Fig. 3.3 E), Stat-3 staining was definite in PRs and strong staining was present in
Müller glia projections and nuclei in Rds+/- retinas (Fig. 3.3 F). Müller glia nuclei were identified using
p27kip1, which co-localized with Stat-3 (Fig. 3.3 G,H). Strong pStat-3 staining was observed in Müller
glia nuclei while PR nuclei were weakly stained. Interestingly, not all Müller glia nuclei in Rds+/-
retinas exhibited pStat-3 staining (Fig. 3.3 J), although all the Müller glia stained for Stat-3; instead
“hotspots” of pStat-3 expression were evident; these pStat-3 “hotspots” co-localized with a subset
of p27kip1 positive Müller glia (Fig. 3.3 K, L) (C. Jiang, McInnes Lab, unpublished). Even when
overexposed, only a few nuclei adjacent to the “hotspots” were weakly stained, but the level of
pStat-3 in the majority of Rds+/- Müller nuclei was below the level of detection (data not shown);
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Figure 3.3: Increased Osmr, Stat3 and pStat3 staining in Müller glia of Rds+/- retinas Representative confocal micrographs of retinal expression of Osmr, Stat-3 and pStat-3 in 7 week-old Rds+/- (B-D, F-H, J-L) and Rds+/+ animals (A,E,I). A, Antibodies recognizing Osmr show diffused staining in the inner plexiform layer (IPL) of Rds+/+ retinas. B, increased staining in Müller cell projections (arrow head) as identified by Gfap (C). D, Gfap and Osmr are co-localized. E, Stat-3 is below the level of detection in Rds+/+ retinas. F, Increased Stat-3 staining in the inner nuclear layer (INL) and IPL, characteristic of Müller glia nuclei (arrow) and projections (arrowhead); staining was also observed in the outer nuclear layer (ONL). G, A marker of Müller glia nuclei, p27kip1, co-localized with Stat-3 (H). I, pStat-3 is below the level of detection in Rds+/+ retinas. J, pStat-3 expression in a subset of cells in the INL (arrow), identified as Müller glia (K), (L). M, No Osmr staining was observed in Osmr-/- retinas. N, no staining was observed in the secondary only negative control. Scale bars indicate 10µm (A-D).
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pStat-3 staining was not observed in any part of wild-type retinas (Fig. 3.3 I). No staining was
observed when immunofluorescence was performed omitting the primary antibody (Fig. 3.3 N).
These results demonstrate that the Osmr and Stat-3 proteins are up-regulated in Rds+/- Müller glia
while pStat-3 is up-regulated in a subset of Müller glia nuclei of Rds+/- retinas.
Lif is a secreted cytokine with autocrine and paracrine capabilities (Cheng and Patterson, 1997;
Nakanishi et al., 2007). To determine which cells produce the Lif transcript in wild-type and Rds+/-
retinas, in situ hybridization using a probe specific to ~1 kb of the 3’ UTR of Lif was performed. All
nuclear layers expressed the Lif mRNA in both wild-type and Rds+/- retinas; however, Lif mRNA
expression was increased by the greatest magnitude in the INL of Rds+/- compared to wild-type
retinas (Fig. 3.4 A,B). No staining was observed when the sense negative control probe was used
(Fig. 3.4 C). To quantify the magnitude of the increase in Lif mRNA expression, I used laser capture
microdissection followed by qPCR. Total cDNA derived from the INL and ONL was tested with a
marker specific to each layer to establish the degree of enrichment achieved using laser capture. Chx-
10, a transcript specific to the inner nuclear layer (Liu et al., 1994) exhibited a 21- fold average
enrichment in INL samples compared to the ONL samples (Fig. 3.5 C), whereas rhodopsin, a PR-
specific marker (Molday and Molday, 1979), was 36-fold enriched in the ONL samples compared to
the INL samples (Fig. 3.5 D), suggesting that the laser capture was effective in isolating material
specific to each of the INL and ONL. Consistent with the in situ hybridization results, Lif was found
to be 4.1-fold up-regulated in Rds+/- compared to wild-type INL samples (p<0.04), while it was not
differentially expressed in Rds+/- compared to wild-type ONL samples (Fig. 3.5 E).
Immunofluorescence analysis was then employed to identify the retinal cell types that expressed
increased amounts of Lif in the Rds+/- retinas. Increased Lif protein staining was observed in Müller
glia projections and PRs of Rds+/- compared to wild-type retinas (Fig. 3.5 F,G); Müller cell
projections were identified by Gfap, which co-localized with Lif (Fig. 3.5 H,I). In summary, my
expression analyses suggest that many cell types produce Lif in wild-type retinas, but that the
greatest increase in Lif expression in Rds+/- retinas occurred in Müller glia.
To identify the cell type(s) that express C/EBPδ transcript, I used laser capture followed by
qPCR analysis. A significant increase in the C/EBPδ transcript was present in the ONL of Rds+/-
retinas, with a 6.2-fold increase in transcript levels (p<0.04), while it was not significantly
differentially expressed in the INL of Rds+/- compared to wild-type retinas (Fig. 3.5 F). Interestingly,
the basal levels of C/EBPδ transcript in the wild-type INL was ~3 fold greater compared to the
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Figure 3.4: Lif and C/EBPδ are up-regulated in several cell types in the retinas of 7-week
old Rds+/- mice
A, Lif mRNA is present in the outer nuclear layer (ONL), inner nuclear layer (INL) and ganglion cell layer (GCL) of Rds+/+ retinas and is up-regulated in the ONL and INL of Rds+/- retinas (B). C, No staining was observed when a sense negative control was used for either Lif. F, Antibodies recognizing Lif display staining in Müller cell projections of Rds+/+ retinas; increased staining was observed in the ONL and Müller cell projections (arrowhead) in Rds+/- retinas (G), which were identified using Gfap, a Müller cell marker (H). I, Co-localization of Gfap and Lif. J, No staining was observed in the secondary-only negative control. Scale bars indicate 10µm (A-J)
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Figure 3.5: Lif up-regulation occurs predominantly in the inner nuclear layer (INL), while C/EBPδ up-regulation is observed mainly in the outer nuclear layer (ONL) Laser capture microdissection followed by qPCR was used to quantify Lif and C/EBPδ transcripts in Rds+/- and Rds+/+ retinas. A, Hematoxylin and Eosin stained frozen section prior to laser capture microdissection with intact ONL and INL. B, Following laser capture, cDNA from the ONL and INL were isolated. C, Chx10, a marker of INL cells was present at 37.5 units+/-2.4 (n=3) in wild-type INL cells and 29.0 units+/-3.2 (n=3) in Rds+/- INL cells, while only present at 1.2 units+/-0.42 (n=3) in wild-type ONL cells and 2.0 units+/-0.36 (n=3) in Rds+/- ONL cells. D, Rhodopsin, a marker of ONL cells was present at 138.3 units +/-49.5 (n=3) in wild-type ONL cells and 105.6 units+/-21.1 (n=3) in Rds+/- ONL cells, while only present at 4.9 units+/-2.5 (n=3) in wild-type INL cells and 2.0 units+/-1.3 (n=3) in Rds+/- INL cells. E, Lif was up-regulated 4.1 fold (p<0.04) in the INL of Rds+/- retinas (6.6 units+/-1.5 (n=3) in Rds+/+ INL cells compared to 26.7 units+/-8.4 (n=3) in Rds+/- INL cells , whereas Lif was not differentially expressed between wild-type and Rds+/- ONL cells. F, C/EBPδ was up-regulated 6.2 fold (p<0.04) in the ONL of Rds+/- retinas (1.1 units+/-0.11 in Rds+/+ ONL cells compared to 6.9 units+/-2.5 in Rds+/- ONL cells; n=3), whereas C/EBPδ was not differentially expressed between wild-type and Rds+/- retinas. All units values were followed by +/- standard error of the mean and all p values were calculated using a t test.
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ONL (Fig. 3.5 F), suggesting that C/EBPδ is normally expressed at low levels in wild-type ONL
cells.
Treatment with IL-6 cytokines have been shown to slow PR cell death in rodent IPD and
light-mediated damage models, concomitant with an increase in pStat-3 levels (LaVail et al., 1992;
Song et al., 2003; Ueki et al., 2008). To determine if endogenous increases mediate a survival response
in mutant retinas, I generated mice carrying both an IPD mutation and loss-of-function mutations
of Osmr, Lif or C/EBPδ. Osmr loss-of-function decreased PR cell survival in two models of IPD: 4-
month old Rds+/-;Osmr-/- retinas had 12.5% fewer PRs compared to Rds+/-;Osmr+/+ (n=9, p<0.05)
(Fig. 3.6 A), whereas 31-day old P347S;Osmr-/- retinas had 13.5% fewer PRs (n=6, p<0.01) compared
to P347S;Osmr+/+ retinas (Fig. 3.6 B). No difference in PR number was observed between 4-month
old Osmr+/+ and Osmr-/- retinas (n=3) (Fig. 3.6 C). These results suggest that the endogenous up-
regulation of the Osmr that occurs in IPD retinas is protective, supporting my hypothesis that the
endogenous activation of IL-6 cytokine pathways in mutant retinas is a survival response. These
results also demonstrate that the loss of the Osmr, in a wild-type background, has no effect on PR
survival.
It has been suggested that the rescue of mutant PRs by cytokine treatment is mediated by
the increased expression of pStat-3 in Müller glia and PRs (LaVail et al., 1992; Song et al., 2003; Ueki
et al., 2008). To determine if pStat-3 levels were affected by the loss of the Osmr, immunoblot
analysis was performed. Surprisingly, there was no significant change in the levels of either Stat-3 or
pStat-3 in Rds+/-;Osmr-/- compared to Rds+/-;Osmr+/+ retinas (Fig. 3.6 D). Similarly, there was no
difference in pERK-1,2 or ERK-1,2 levels in Rds+/-;Osmr-/- compared to Rds+/-;Osmr+/+ retinas (Fig.
3.6 D), suggesting that Osmr-mediated protection is occurring independently of Stat-3 or ERK-1,2.
In contrast to the protective effects of Osmr in two models of IPD, we were surprised to
observe that Lif , a cytokine known to activate Stat-3, was pathogenic in the Rd1-/- model of IPD.
There was a 14% increase in PR cell number in PN13 Rd1-/-;Lif -/- compared to Rd1-/-;Lif +/+ retinas
(n=6, p<0.003) (Fig. 3.7 A); there was no difference in PR cell number between PN13 Lif- /-
compared to Lif +/+ retinas (Fig. 3.7 B). To determine if pStat-3 and total Stat-3 levels were altered as
a result of the loss of Lif, immunoblot analysis was performed. We identified a 1.7 fold decrease in
pStat-3 in PN13 Rd1-/-;Lif -/- compared to Rd1-/-;Lif +/+ retinas, while no significant difference in Stat-
3 levels were observed (Fig. 3.7 C). Similarly, C/EBPδ, a gene down-stream of Stat-3 (Hutt et al.,
2000), was also found to be pathogenic. C/EBPδ loss-of-function increased mutant PR survival:
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Figure 3.6: In two models of PR degeneration, Osmr is protective in a pStat-3 independent mechanism Representative toluidine blue stained retinal sections (A-C) and immunoblot analysis comparing the total and phosphorylated forms of Stat-3 and ERK-1,2 in Rds+/-;Osmr+/+ and Rds+/-;Osmr -/- retinas (D). A, The outer nuclear layer (ONL) of Rds+/-;Osmr -/- is 12.5% thinner compared to Rds+/-;Osmr+/+ retinas harvested at four months-of-age (Rds+/-;Osmr -/- ONL is 23.8µm +/-.88µm (n=9) versus Rds+/-;Osmr+/+ ONL which is 28.0µm +/-1.6µm (n=9); p<.05). B, The ONL of P347S;Osmr -/- is 13.5% thinner compared to P347S;Osmr+/+ retinas harvested at 31 days of age (P347S;Osmr -/- ONL is 13.9µm +/-0.25µm (n=6) compared to P327S;Osmr+/+ ONL, which is 16.0µm +/-0.67µm (n=6); p<0.01). C, There was no significant difference in ONL thickness between Osmr+/+ compared to Osmr -/- retinas harvested at 4 months of age. Scale bars indicate 25µm. All values were mean ONL thickness+/- standard error of the mean; p values were computed using a two-tailed t test. D, Representative immunoblot showing that the levels of pStat-3, Stat-3, pERK-1,2, and ERK-1,2 are not significantly different in retinal lysates harvested from 7-week old Rds+/-;Osmr+/+ and Rds+/-;Osmr -
/- mice (n=4).
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Figure 3.7: Lif is pathogenic in the Rd1 -/- model of PR degeneration Representative toluidine blue stained retinal sections (A,B) and immunoblot analysis comparing the total and phosphorylated forms of Stat-3 in Rd1-/-;Lif +/+ and Rd1-/-;Lif -/- retinas (C). A, The outer nuclear layer (ONL) of Rd1-/-;Lif -/- is 14.0% thicker compared to Rd1-/-;Lif +/+ retinas harvested at PN13 (Rd1-/-;Lif -/- ONL is 33.2µm +/-.0.60µm (n=7) versus Rd1-/-;Lif +/+ ONL which is 28.5µm +/-0.59µm (n=6); p<0.003). B, There was no significant difference in ONL thickness between Lif +/+ compared to Lif -/- retinas harvested at PN13. Scale bars indicate 25µm. All values were mean ONL thickness+/- standard error of the mean; p values were computed using an ANOVA. C, Immunoblot showing that the levels of pStat-3 decreases in Rd1-/-;Lif -/- compared to Rd1-/-;Lif +/+ retinas. Densitometry analysis revealed: a 1.7-fold down-regulation of pStat-3 in Rd1-/-;Lif -/- (n=4, 18730 pixels +/- 2227) compared to Rd1-/-;Lif +/+ (n=4, 33318 pixels +/- 2744; p<0.05). There was no significant decrease in Stat-3 levels. p values were computed using a paired two-tailed t test.
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8-month old Rds+/-;C/EBPδ -/- retinas had 18% more PRs compared to Rds+/-;C/EBPδ+/+ (n=5,
p<0.005). The loss of C/EBPδ in a wild-type background had no effect on the ONL thickness (Fig.
3.8 B).
Discussion
We have identified several up-regulated IL-6 cytokines, receptors and downstream pathway
members in three models of IPD. For most of these molecules, the magnitude of the up-regulation
correlated with the rate of cell death exhibited by each model. For example, the Osmr transcript was
up-regulated 5.6-fold in the faster Rd1-/- model compared to a 3.4-fold increase in the intermediate
P347S model, and a 2.6-fold up-regulation in the Rds+/- model, the slowest degeneration of these
three models. These findings are consistent with the IL-6 pathway functioning in response to cell
stress and may represent a global response to IPD. One notable exception to this trend was the
oncostatin ligand, which was up-regulated in the Rds+/- and P347S but not the Rd1-/- model of IPD.
This result was unexpected because this cytokine can only bind Osmr (Heinrich et al., 2003), which
was up-regulated in all three models examined. If the abundance of the Osmr is rate-limiting in the
mutant retina, then increased signalling could occur due to the increase in receptor abundance,
without an increase in the cytokine. However, other IL-6 receptors do not appear to be rate-limiting,
as illustrated by the increase in Stat-3 phosphorylation upon cytokine addition (Peterson et al., 2000;
Song et al., 2003). Alternatively, Osmr signalling might only occur in a subset of IPD models.
The expression of all IL-6 cytokines and receptors was assayed in Rds+/- and wild-type
retinas. Of the 8 cytokines and six receptors in the IL-6 family, only Osm, Lif, Osmr, IL-6r and gp130
transcripts were identified as up-regulated, suggesting a specific IL-6 response rather than a general
up-regulation of the whole family of IL-6 cytokines and receptors in the mutant retina. The
observation that the Osm transcript was up-regulated in the Rds+/-and P347S but not the Rd1-/- model
of IPD, is consistent with another report which concluded that different models of IPD can
differentially activate specific members of the IL-6 family of cytokines/receptors; for example, Lif
and Clc mRNA were up-regulated in the Rd1-/- and Vpp-/- models of IPD, while Cntf was found to
be up-regulated only in the Rd1-/- model (Samardzija et al., 2006). Taken together with our findings,
Lif and Osmr up-regulation may be a general response to IPD, while other cytokines such as Osm,
Clc, and Cntf may be up-regulated in a subset of IPD models.
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Figure 3.8: C/EBPδ is pathogenic in the Rds+/- model of PR degeneration
Representative toluidine blue stained retinal sections from 8-month old animals. A, The ONL is 18.2% thicker in Rds+/-;C/EBPδ -/- compared to Rds+/-;C/EBPδ+/+ mice (Rds+/-;C/EBPδ -/- ONL is 28µm+/-1.7 compared to Rds+/-;C/EBPδ+/+ ONL which is 22.9µm +/-0.38; (n=5, p<0.006). B, The ONL is the same thickness in C/EBPδ+/+ and C/EBPδ -/- animals (n=3). Scale bars indicate 10µm. All values were mean ONL thickness+/- standard error of the mean; p values were computed using a two-tailed t test.
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The up-regulation of Osm, Lif and the Osmr transcripts in Rds+/- retinas suggested that the
signalling pathways downstream of the IL-6 cytokines may be more active in IPDs. To investigate
this possibility, I examined the activity of the ERK-1,2 and STAT pathways, the two major pathways
downstream of the IL-6 family of cytokines (Kamimura et al., 2003). On examining the
phosphorylation state of Stat-1, Stat-3 and ERK-1,2, I found that only pStat-3 was significantly up-
regulated. Interestingly, ERK-1,2 has been shown to be up-activated in Cntf-treated retinas
(Peterson et al., 2000) and in light-induced PR degeneration but not in IPD models (Samardzija et
al., 2006). Similarly, pStat-1, a pro-apoptotic STAT family member, has been identified as up-
activated in retinas after light-mediated damage, but not in IPD models (Samardzija et al., 2006). My
results are consistent with these other reports and suggest that Stat-3 may have a key role in
inherited PR degeneration, while Stat-1 and ERK-1,2 may respond to light-mediated damage but not
to genetically mediated damage.
Phosphorylated Stat-3 has been shown to be up-regulated in Müller glia of retinas treated
with IL-6 cytokines. Wild-type animals treated with Cntf (Wang et al., 2002), axokine (a Cntf analog)
(Peterson et al., 2000), or Lif (Ueki et al., 2008) and transgenic rats carrying the rhodopsin mutation
S334ter treated with cardiotrophin-1 (Song et al., 2003) all displayed increased Stat-3 and pStat-3 in
Müller glia. Only Lif treatment resulted in increased Stat-3 and pStat-3 staining in wild-type PRs
(Ueki et al., 2008). While a previous report has shown increased Stat-3 and pStat-3 protein in the
retinas of the Rd1-/- and Vpp-/- mouse models of IPD (Samardzija et al., 2006), the localization of
Stat-3 or pStat-3 has not been reported in any IPD model. We found that the endogenous protein
levels of Stat-3 and pStat-3 were up-regulated in the Müller glia of Rds+/- retinas. Interestingly, in
Rds+/- retinas, the increase in pStat-3 was detected predominantly in a subset of Müller glia, creating
foci or “hotspots” of pStat-3 that were situated at fairly regular intervals throughout the retina (C.
Jiang, McInnes Lab, unpublished). In contrast, Stat-3 was increased uniformly throughout all Müller
glia of the Rds+/- retinas. The origin of these pStat-3 “hotspots” is not currently known. One
hypothesis is that glia in close proximity to a dying neuron might respond through Stat-3
phosphorylation. Immunofluorescence analysis suggests Stat-3 might also be present in PRs. The
presence of the Osmr in Müller glia suggests that signalling from the Osmr can activate Stat-3 in
these cells; the lack of Osmr staining in PRs suggests that any PR specific activation of Stat-3 would
have to occur through an Osmr-independent mechanism. Since mutant PRs are protected by
cytokine treatment, Wahlin et al. suggested that the up-regulation of cytokine receptors in PRs as a
possible mechanism accounting for the PR-specific protective effects of neurotrophin treatment
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(Wahlin et al., 2001). Our results do not support this hypothesis for the Osmr, which was not
detected in PRs. Instead, our results support the notion of the Müller cell hypothesis, which
proposes that Muller glia activation represents a survival response for mutant PRs (Harada et al.,
2000; Zack, 2000).
Lif protein was identified as up-regulated in Müller glia and PRs; Lif mRNA was also present
in all inner nuclear layer cells and ganglion cells. One interpretation of these results is that although
multiple cell types are capable of producing Lif, the receptor is present predominantly on Müller glia
and PRs. Treatment with Lif increases pStat-3 in both Müller glia and PRs, supporting the
hypothesis that Lif can bind to receptors on those cell types (LaVail et al., 1998; Ueki et al., 2008);
however, there is no published data on the localization of the Lifr in the retina. C/EBPδ is a
transcription factor known to be activated by pStat-3 (Yamada et al., 1997b; Hutt et al., 2000).
Consequently, I initially interpreted the microarray and qPCR results which indicated that C/EBPδ
was up-regulated in the Rds+/-, P347S and Rd1-/- retinas as being a result of Osmr activation. I
therefore extrapolated that the increase in C/EBPδ mRNA expression would be in Müller glia.
Although I did identify C/EBPδ transcript expression in the inner nuclear layer (which includes
Müller glia nuclei) of Rds+/- and wild-type retinas, I observed a significant increase in
C/EBPδ mRNA expression in the PRs of Rds+/- mice using laser capture microdissection and
qPCR. These results suggest that the up-regulation of C/EBPδ transcript in PRs must be mediated
by another cytokine receptor, since I was unable to detect the Osmr in PRs (Fig. 3 A). Thus the
putative IL-6 pathway in the retina would appear to be more complex than the simple linear
pathway proposed in Chapter 2, because 1) the loss of the Osmr does not affect the level of pStat-3
expression; 2) C/EBPδ transcript was up-regulated only in the ONL of Rds+/- retinas, whereas
Osmr up-regulation was only detected in Muller cells and astrocytes of Rds+/- retinas. Rather, it
would appear that the level of pStat-3 is maintained by IL-6 receptors other than Osmr, and/or that
pStat-3 regulation varies in different retinal cell types.
The absence of the Osmr in the Rds+/- and P347S models of IPD led to increased PR cell
death, suggesting that the Osmr is required for an optimal survival response in retinas with PR
mutations. Many reports have shown that IL-6 cytokines are protective in both light-induced and
IPD retinas; pStat-3 and/or pERK-1,2 have been hypothesized as possible mediators of PR
protection (Liu et al., 1998; Song et al., 2003; Samardzija et al., 2006; Ueki et al., 2008). Interestingly,
we did not observe any changes in either pStat-3 or pERK-1,2 levels in Rds+/-;Osmr-/- compared to
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Rds+/-;Osmr+/+ retinas, suggesting that the Osmr-mediated protection may not be mediated solely or
predominantly by pStat-3 and pERK-1,2. Further work is required to determine whether Stat-
3/pStat-3 are essential to the endogenous retinal responses to the presence of a PR mutation. If they
are, then the identification of downstream targets of pStat-3 will provide valuable insight into the
molecular mechanisms that underlie the protective effect.
The failure of pStat-3 levels to decrease upon loss of the Osmr could be interpreted in
several ways. First, if signalling through the Osmr is not the primary pathway resulting in Stat-3
phosphorylation, then the loss of the Osmr would have a negligible affect on pStat-3 levels. Second,
although the overall retinal abundance of pStat-3 did not change in the absence of the Osmr, its
levels in specific retinal cells may have decreased, but was not detectable by immunoblotting. Third,
a compensatory system might exist such that other factors are up-regulated in the absence of the
Osmr. The transcript levels of Lif, Lifr, Cntf, and Ct-1 were examined and were not up-regulated in
response to the loss of the Osmr in Rds+/- retinas, suggesting that these IL-6 cytokines are not
participating in any compensatory response that sustains the level of pStat-3.
In contrast to the protective effects of the Osmr in Rds+/- and P347S retinas, we identified
Lif and C/EBPδ as essential components of the pathogenic consequences of a PR carrying an IPD
mutation. A pro-apoptotic role for Lif and C/EBPδ has been reported during mammary
development; for example, during mammary glad involution, Lif signalling results in Stat-3
phosphorylation followed by C/EBPδ up-regulation, which then leads to the activation of pro-
apoptotic genes and the suppression of anti-apoptotic genes (Thangaraju et al., 2005; Clarkson et al.,
2006). C/EBPδ null allele mice also exhibit mammary gland hyperplasia, consistent with reduced
developmental apoptosis (Gigliotti et al., 2003). The fact that the Osmr was up-regulated in Müller
glia while C/EBPδ was predominantly up-regulated in PRs may account for the apparently
contradictory roles of these two members of the same pathway. We hypothesize that C/EBPδ may
be required for the pathogenic effect of PR mutations. In contrast, the up-regulation of the Osmr in
Müller glia mediates a protective response.
The finding that Lif is pathogenic in the Rd1-/- model of IPD was unexpected given the
protective affects of Lif treatment in other IPD models and in light-mediated PR cell death (LaVail
et al., 1998; Ueki et al., 2008). Furthermore, pStat-3 levels were reduced 1.7-fold in Rd1-/-;Lif-/-
compared to Rd1-/-;Lif+/+ retinas, suggesting that maintenance of pStat-3 is not essential for
protection, and/or that, paradoxically in this model, the reduction of pStat-3 was actually protective.
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One or more alternative mechanisms could account for these results. First, Lif has been established
as an important cytokine involved in PR differentiation during development (Elliott et al., 2006).
Although the total number and histological appearance of PRs was not different between Lif-/- and
Lif+/+ retinas, the lack of Lif during development may have somehow modified the response to the
Rd1 mutation in Rd1-/-;Lif-/- retinas, changing the rate of cell death. Second, Lif may actually be
pathogenic in some models of IPD. Lif treatment is protective in the RCS rat, and two rhodopsin
transgenic rat models (Matthew LaVail, personal communication), whereas Rd1-/- retinas treated with
Lif did not show any PR protection (LaVail et al., 1998). Since the treatment involved only a single
injection of Lif, it is possible that a more constant albeit endogenous exposure to Lif may be
pathogenic in the Rd1-/- model. Since pStat-3 has been shown to be activated in PRs in response to
Lif injection (Ueki et al., 2008), Lif may be capable of activating C/EBPδ in PRs, resulting in PR cell
death. Conversely, the lack of Lif may decrease C/EBPδ levels, leading to increased PR survival.
The finding that Lif and C/EBPδ are pathogenic in Rd1-/- and Rds+/- retinas, respectively,
may have important therapeutic implications. Animal studies indicating Cntf treatment is effective in
delaying PR cell death in animal IPD models led to further investigations of Cntf as a possible
therapy in humans (Sieving et al., 2006). While the efficacy of Cntf treatment in humans remains to
be determined, the possible activation of C/EBPδ in PRs- a potentially pathogenic response- should
also be investigated. The presence of the Cntf receptor on PRs has been identified by one study
(Valter et al., 2003), consistent with the possibility that Cntf treatment could increase C/EBPδ levels
in mutant PRs. The long-term activation of C/EBPδ may have harmful effects and partially mitigate
the protective effects of Cntf mediated through other cells such as Müller glia. If further studies
support a pathogenic role for C/EBPδ in mutant PRs, then the use of reagents such as PR-specific
siRNAs to C/EBPδ, may prolong the survival of mutant PRs. Similarly, the finding that long-term
exposure to increased levels of Lif may be pathogenic has obvious implications for the use of Lif as
a treatment. Further studies are required to confirm the pathogenic role for Lif and determine
whether its pathogenicity is unique to the Rd1-/- model.
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4 General discussion and future directions
4.1 Significance of findings
Inherited PR degenerations result from mutations in many different functional categories of
genes, but it is unlikely that each mutation triggers a unique pathway that results in cell death.
Historically, research on IPDs has focused on understanding the normal function of the proteins
encoded by IPD genes; however, with few exceptions, this approach has not led to significant
insights into why mutant PRs die. In this thesis I have described an unbiased, whole-genome
approach to identify transcripts with altered expression in the retinas of animals with IPDs. I
focused my attention on a subset of the differentially expressed genes comprising a putative IL-6
signalling pathway because i) they were among the most significantly altered in their expression; ii)
studies by others had shown that some of these genes displayed increased expression that correlated
with- but had not been mechanistically linked to- a protective response initiated by IL-6 cytokines,
suggesting that the responses of these genes I identified might also mediate an endogenous survival
response to the presence of a PR mutation; and iii) many of the differentially expressed genes I
found had also been identified in studies by other groups examining altered gene expression in a
wide variety of IPD and light-damaged models. That these transcripts are up-regulated in a range of
genetic and non-genetic insults to the retina is consistent with my hypothesis that they represent a
general response that are not related to the specific biochemical abnormalities conferred by the
disruption of any particular IPD protein. In none of these other studies, however, was the
pathogenic significance of these changes in gene expression investigated. In my work, in contrast, I
took advantage of the availability of mouse strains carrying loss-of-function alleles of several of the
genes I found to be differentially expressed in IPD models, to examine the role of these genes in the
pathogenesis of PR degeneration.
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In the second chapter of this thesis, I described the microarray and quantitative PCR (qPCR)
experiments in which I examined the transcriptomes of the Rom1-/- and Rds+/- murine models of
IPD. I sampled the mRNA population of those models at times when >90% of PRs were present in
each model. My studies are one of the first to have identified differentially expressed transcripts in
IPD retinas at time-points before significant PR degeneration has occurred. The differentially
expressed genes represent early responses to a pathogenic mutation; these responses may be
pathogenic, protective or neutral in their relationship to PR death.
I identified up-regulated transcripts in the retinas of Rds+/- mice that I hypothesized to be
participating in a common IL-6 signalling pathway, including the mRNAs of Lif, Osm, Osmr, Stat-3,
C/EBPδ, Socs3, and α2macroglobulin. Since treatment with IL-6 cytokines is neuroprotective in
light-damaged and IPD models, as indicated above, my working hypothesis was that IL-6 signalling
in the IPD retina was also a protective response (LaVail et al., 1992; LaVail et al., 1998).
In chapter three, I further examined the role of members of the putative IL-6 pathway in
IPDs. After examining the expression levels of all eight IL-6 cytokines and six IL-6 receptors in the
retinas of Rds+/- mice, I determined that Lif, Osmr and gp130 were also differentially expressed in
the Rd1-/- and P347S models of IPD, suggesting that an IL-6 cytokine response may occur generally
in IPDs. The observation that phosphorylated Stat-3 (pStat-3) was increased in Rds+/- retinas
suggests that an IL-6 pathway is up-regulated in mutant retinas. Although increased pStat-3 had
been identified in light-damaged retinas and wild-type retinas treated with IL-6 cytokines, my work is
one of the first studies to identify increased pStat-3 in IPD retinas, and the first to establish the
identity of the cell types expressing Lif, Osmr, Stat-3, pStat-3, and C/EBPδ in IPD retinas. While
Osmr was localized exclusively to Müller glia and astrocytes, Stat-3, pStat-3, Lif and C/EBPδ were
expressed in multiple cell types, including PRs, in the Rds+/- retina. The observation that Stat-3 and
its downstream target, C/EBPδ (Yamada et al., 1997a), are both expressed in PRs, suggests that an
IL-6 receptor other than Osmr must mediate the activation of Stat-3 in PRs. My work provides the
first indication that the IL-6 signalling responses in IPD retinas may be more complicated than a
simple linear model in which all components are functioning in a single cell type.
My use of genetic analyses to determine if the loss of Lif, Osmr or C/EBPδ expression
would modify the rate of PR death in IPD models is also novel. As predicted by the protective
effect of IL-6 cytokines on IPDs, the Osmr loss-of-function allele in the Rds+/- and P347S models of
IPD increased PR death, demonstrating that the presence of the Osmr is required for a protective
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response. Unexpectedly, pStat-3 levels did not decrease in Rds+/-;Osmr-/- retinas (i.e. in the absence of
Osmr), suggesting either that pStat-3 is not solely regulated by Osmr in the mutant retina, or that the
protective role of Osmr does not require a change in the steady-state level of pStat-3 in the whole
IPD retina. Given that C/EBPδ is a downstream target of IL-6 signalling (Hutt et al., 2000), I
originally predicted that C/EBPδ would be a protective molecule. However, my genetic analyses
suggest that C/EBPδ plays a previously unrecognized pathogenic role in the IPD retina, which is
consistent with its pro-apoptotic role during mammary gland development (Thangaraju et al., 2005).
Thus, we propose that C/EBPδ signalling is pathogenic when expressed in mutant PRs and may be
a novel therapeutic target. My finding that the Lif loss-of-function allele in Rd1-/- retinas decreases
the expression of pStat-3 and increases the number of surviving mutant PRs was also unexpected
given that treatment with Lif is protective in light-damage and several IPD models (Mathew Lavail,
personal communication and (Ueki et al., 2008)). Thus, Lif may be pathogenic in the Rd1-/- model.
Alternatively, a secondary affect of Lif loss-of-function in IPD retinas may account for the increased
PR cell number. These results add new insight into IL-6 signalling in the retina. They may also have
therapeutic implications, since an IL-6 cytokine (Cntf) is being tested as a therapy in human retinitis
pigmentosa patients (Sieving et al., 2006).
4.2 Identifying the mechanism(s) of IL-6 mediated photoreceptor protection
The mechanism by which IL-6 cytokines protect PRs is not known, although the correlation
between activation of Stat-3 expression following IL-6 cytokine administration, and the protective
effect of IL-6 cytokines, suggests that pStat-3 mediated signaling may be critical. (Kamimura et al.,
2003; Samardzija et al., 2006). The work presented here and by Samardzija et al. show that Stat-3
activation occurs in many models of IPD, which may be a protective response to a pathogenic
mutation (Peterson et al., 2000; Ueki et al., 2008). I demonstrated that Osmr loss-of-function
increased the rate of PR death in the Rds+/- model, although the levels of both pStat-3 and pERK-
1,2, the two major mediators of IL-6 signalling, remained unchanged in the absence of Osmr. This
result does not directly refute the putative protective role of pStat-3 or pERK-1,2, but it does raise
the possibility that the Osmr may activate other pathways involved in protecting PRs. One strategy
to identify pathways downstream of Osmr would be to compare the retinal transcriptomes of Rds+/-
transcripts that may be involved in a non-Stat-3 and non-ERK-1,2 pathway downstream of Osmr.
Other molecular components of the IL-6 pathway could also be identified by examining the
retinal transcriptome after IL-6 cytokine treatment of the mutant retina, assuming that cytokine-
mediated PR rescue requires de novo transcription. Treatment with Cntf has been shown to protect
PRs in IPD models; however at high doses, PR morphology and function is abnormal (Liang et al.,
2001). A recent examination of the transcriptional effects of sustained high doses of Cntf in the
murine retina was used to gain insight into Cntf-induced toxicity (Rhee et al., 2007). Rhee et al.
compared the retinal transcriptome of Rds+/- mice treated with adeno-associated virus (rAAV)
expressing Cntf to the retinal mRNA population of untreated Rds+/- mice (Rhee et al., 2007). Among
the 50 significantly differentially expressed genes identified, several members of the
phototransduction cascade were down-regulated, which may account for the reduced ERG activity
in rAAV-Cntf treated retinas (Rhee et al., 2007). Since Cntf slows mutant PR death, the microarray
analysis comparing rAAV-Cntf treated Rds+/- retinas and untreated Rds+/- controls may have also
identified candidate neuroprotective genes. Stat-3 and ERK-1,2, were both up-regulated in treated
retinas, while two pro-apoptotic genes, programmed cell death protein 4 (pdcd4) and programmed
cell death protein 7 (pdcd7), were down-regulated ~2-fold (Rhee et al., 2007). These findings are
consistent with the deceased PR death observed in Cntf-treated retinas (Rhee et al., 2007). While
pdcd7 is a tumour suppressor gene involved in the repression of cell cycle proteins (Goke et al.,
2004), pdcd4 is a pro-apoptotic gene involved in inducing cell death in T-cells (Park et al., 1999).
Further characterization will be required before the role of these genes in the Cntf protection of
mutant PRs can be understood. One drawback to the design of this experiment for the identification
of genes involved in PR protection is the toxic affect of high doses of Cntf on PRs, as indicated by
the reduced ERGs of treated PRs. A better experimental design to identify protective genes would
be to treat Rds+/- retinas with a lower dose of Cntf, using, for example, encapsulated cell technology
to provide prolonged Cntf exposure, and to repeat the microarray experiment on retinas from
treated and untreated Rds+/- animals. The identification of differentially expressed genes may identify
the molecules that mediate neuroprotection following endogenous increases in Cntf expression or
exogenous Cntf treatment.
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4.3 The mode of IL-6 signalling: direct or indirect action on photoreceptors
The identification of the cell types that respond to IL-6 cytokines is important in order to
understand the mechanism of IL-6 cytokine mediated PR protection. The Müller cell hypothesis
suggests that Müller glia respond to exogenous or endogenous signals to protect PRs from cell death
(Zack, 2000). Data presented in this thesis and by Ueki et al. suggest that IL-6 cytokines may directly
activate downstream responses in PRs (Ueki et al., 2008). There are at least four possible types of
retinal cell responses following IL-6 cytokine administration in IPDs: 1) PRs may respond directly to
IL-6 cytokines; 2) Müller glia may respond to IL-6 signalling and in turn protect PRs; 3) both cell
types may respond to IL-6 signalling; 4) other retinal cell types may respond, although to date there
is no evidence bearing on this possibility.
Consistent with the Müller cell hypothesis, treatment with Cntf or Lif results in the up-
regulation of pStat-3 predominantly in Müller glia concomitant with PR cell protection in light-
damaged or IPD models (Peterson et al., 2000; Ueki et al., 2008). Harada et al. identified several
molecules involved in Müller glia-PR signalling in the light-damage model (Harada et al., 2000).
Müller cells respond to nerve growth factor (NGF) or neurotrophin-3 (NT-3) by modulating the
release of basic fibroblast growth factor (bFGF); increased NGF correlated with decreased bFGF
release by Müller cells and increased PR cell death, while increased NT-3 correlated with increased
bFGF release by Müller glia and PR rescue (Harada et al., 2000). Some of the work presented in this
thesis is also consistent with a Müller glia response influencing PR survival. For example, I found
that the Osmr was exclusively expressed in Müller glia projections, and the absence of the Osmr
increased the rate of PR death in P347S and Rds+/- retinas. However, I also obtained evidence that an
IL-6 response also occurs in mutant PRs: Lif, Stat-3, pStat-3 and C/EBPδ were all up-regulated in
mutant PRs, suggesting that the direct reponses of mutant PRs may also decrease the rate of PR
death. These findings are consistent with work done by Ueki et al. that showed that Lif treatment of
light-damaged retinas resulted in the up-regulation of pStat-3 in both PRs and Müller cells, and in
PR protection (Ueki et al., 2008).
Several approaches could be taken to examine the importance of direct IL-6 signalling in
protecting PRs. Imaging studies to determine whether IL-6 cytokine receptors are present in PRs
would establish a pre-requisite for IL-6 signalling to occur. The observation that Lif treatment
results in pStat-3 activation in PRs is consistent with the presence of Lif receptors on PRs (Ueki et
al., 2008), but this data should be independently confirmed. I have presented results which suggests
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that the Osmr is not expressed in PRs, while work by others suggests that the Cntf receptor and
gp130 are expressed in both the inner and outer segments of PRs (Beltran et al., 2003; Rhee and
Yang, 2003). However, the expression of the appropriate receptors is only a minimum requirement
for IL-6 signalling to occur in PRs. Several studies have shown that retinas treated with Cntf or
axokine (a Cntf analog) do not exhibit pStat-3 expression in PRs (Peterson et al., 2000; Wang et al.,
2002), despite the presence of all the receptors necessary for Stat-3 signalling to occur.
Consequently, a functional approach is also required to determine if IL-6 signalling occurs in PRs.
A functional approach that directly examines the ability of IL-6 cytokines to activate
downstream signalling molecules in PRs would be strong evidence that IL-6 cytokines act directly on
PRs. Traverso et al. have developed a technique to isolate PRs from a porcine retina and grow them
in culture for two weeks (Traverso et al., 2003). They used this cell culture system to test and
confirm that epidermal growth factor (Egf) and bFGF act directly to protect PRs. Cells grown in the
presence of either factor survived for longer and exhibited increased phosphotyrosine abundance, an
indication of signalling downstream of bFGF or Egf (Traverso et al., 2003). An analogous
experiment could be performed to determine if any of the IL-6 cytokines can affect PR survival in
this assay system, an indication that the cytokine can act directly on PRs. Furthermore, downstream
IL-6 signalling molecules could be directly examined in PRs to determine if cytokine treatment
increased pStat-3 or pERK-1,2 in PRs. If IL-6 cytokines increase the survival of mutant PRs in this
context, and activate Stat-3 or ERK-1,2 signaling molecules, the transcriptomes of isolated wild-type
and mutant PRs could be compared before and after IL-6 treatment, as a first step in dissecting
direct PR-specific responses to cytokine administration.
An in vivo approach could also be taken to determine if IL-6 signalling directly acts on PRs.
Since gp130 is the common co-receptor of all IL-6 cytokine receptors, its presence is necessary to
activate downstream signalling pathways (Heinrich et al., 1998). Transgenic mice expressing a
dominant negative gp130 transgene have been used to assess the role of IL-6 signalling during
antibody production (Kumanogoh et al., 1997). The dominant negative gp130 protein is a truncated
form of the wild-type protein, lacking part of the cytoplasmic domain, which is required for Stat
and Erk binding; expression of the truncated gp130 significantly reduced Stat-3 phosphorylation in
the spleen (Kumanogoh et al., 1997). A dominant negative gp130 transgene could be expressed in
several different retinal cell types of IPD mice. Since IL-6 signalling is required for retinal
development, an inducible truncated gp130 transgene could be created that can be activated in adult
mice (Kistner et al., 1996). If PRs primarily respond to IL-6 signalling, then transgenic mice
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expressing the dominant negative gp130 transgene in PRs alone should be resistant to the protective
effects of IL-6 cytokine treatment after damaging light treatment or in models of IPD. The
analogous experiment could also be performed in transgenic mice expressing the dominant negative
gp130 transgene in Müller glia to determine if IL-6 signalling in these glia contributes to PR
protection. These experiments may determine whether Müller glia, or PRs, or both, underlie the
protection of mutant or light-damaged PRs mediated by IL-6 cytokines.
4.4 Is Stat-3 signalling a protective response?
Many studies have suggested that Stat-3 signalling is protective to light-damaged and IPD
retinas, based on its up-regulation in Müller cells following IL-6 cytokine treatment (Song et al.,
2003; Ueki et al., 2008). Few studies, however, have addressed the fundamental question of whether
the increase in Stat-3 signalling is mechanistically linked to the protective effect of IL-6 cytokines, or
whether it is merely correlative. Samardzija et al. employed the AG490 Jak kinase inhibitor in an
attempt to prevent the phosphorylation of Stat-3 in IPD retinas (Samardzija et al., 2006). Although
AG490 treated IPD retinas did not exhibit any change in the rate of PR death, the result was not
informative because AG490 treated IPD retinas did not exhibit decreased Stat-3 signalling
(Samardzija et al., 2006). However, light-damaged retinas treated with AG490 did exhibit a
significant reduction in both pStat-1 and pStat-3 and increased PR cell survival (Samardzija et al.,
2006). Since Stat-1 is a pro-apoptotic member of the Stat family, whereas Stat-3 is regarded as an
anti-apoptotic protein (Stephanou and Latchman, 2005) and both proteins exhibited decreased
phosphorylation with AG490 treatment, it was not possible to determine if the decrease in pStat-1
or pStat-3 was responsible for the increased PR cell survival. In the experiments presented in this
thesis, I employed a genetic approach to determine if the Osmr or Lif loss-of-function mutants
affected Stat-3 phosphorylation in several IPD models, but these experiments are also correlative in
nature. Consequently, other approaches must be used to ascertain if Stat-3 signalling is protective,
pathogenic or a bystander to cell death.
A genetic approach could be used to determine if Stat-3 directly mediates PR protection. A
Stat-3 floxed allele has already been created to study the effect of Stat-3 in mammary glad
development and could be employed in the retina (Chapman et al., 2000). Since Stat-3 is known to
be expressed in Müller glia and PRs (Ueki et al., 2008), this approach could also be used to identify
not only whether Stat-3 signalling is protective, but in which cell type its expression is required for
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Stat-3 signalling to be protective. An IPD model lacking Stat-3 in PRs could be created. After IL-6
treatment, the rate of cell death could be compared between an IPD model lacking Stat-3 in PRs and
the same IPD model with normal Stat-3 PR expression. If the rate of cell death was affected by the
presence of Stat-3 in PRs, then a direct relationship between PR survival and Stat-3 expression could
be made. A similar methodology could be employed to prevent Stat-3 expression in Müller glia to
determine if Stat-3 expression in Muller glia is responsible for neuroprotection upon IL-6 treatment.
If Stat-3 expression in Müller cells is protective, then another question is to determine what
signal(s) Müller cells are releasing that is mediating PR protection. If both PRs and Müller glia are
capable of responding to IL-6 signalling, resulting in a decrease in PR death, then the role of the
Müller glia signalling may be to amplify the IL-6 cytokine signal. IL-6 cytokines are known to
function as part of a positive feedback loop (Davey et al., 2007). Thus, upon binding an IL-6
cytokine, Müller glia may respond by secreting more of the cytokine, which would increase the
amount of cytokine available to bind to PRs, thereby increasing the neurotrophic effect.
Alternatively, Müller glia may respond to IL-6 cytokines by secreting another protein, which then
protects PRs from cell death. Identifying protective factors produced by Müller glia could be
performed by exposing a Müller cell culture to an IL-6 cytokine and then examining changes in
transcription using microarray technology. A mass spectrometry based method to identify any
proteins that are up-regulated in Müller cell media in response to cytokine treatment might also be
informative, although the sensitivity of this approach may be limited.
4.5 Determining whether C/EBPδ expression in photoreceptors is pathogenic
The finding that C/EBPδ is a pathogenic molecule was unexpected given that its
expression is activated by pStat-3 (Hutt et al., 2000), a putative protective molecule. At least two
possible explanations may account for this data: 1) C/EBPδ expression in the retina may be
regulated by factors in addition to Stat-3 and this alternative regulatory pathway(s) may be
pathogenic in IPDs; 2) in some physiological contexts, perhaps increased pStat-3 expression is,
paradoxically, pathogenic. Quantitative PCR performed on isolated PRs and inner nuclear layer
(INL) cells revealed that C/EBPδ expression is significantly increased in the PRs of Rds+/- vs. wild-
type mice. C/EBPδ has been identified as a pro-apoptotic factor during mammary gland involution
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(Thangaraju et al., 2005), providing a precedent for the pro-apoptotic role I observed in the IPD
retina.
To support the evidence that C/EBPδ is a pathogenic factor in the IPD retina, a transgenic
approach could be used to over-express C/EBPδ moderately in wild-type and IPD PRs. A
significant increase in PR death with increased C/EBPδ PR expression in the IPD and wild-type
retinas would be supportive of the pathogenic role for C/EBPδ in PRs. Alternatively, increased PR
death may only occur in the context of increased C/EBPδ expression in the IPD retina, which
would suggest a pathogenic role for C/EBPδ in the mutant PR. A similar strategy could be
employed to determine if C/EBPδ expression in Müller glia is pathogenic to PRs. My findings
suggest that C/EBPδ is a pathogenic factor in IPDs; however, since Rds+/-;C/EBPδ-/- mice lack
C/EBPδ expression in all cell types, it is not possible to conclude that the PR expression of
C/EBPδ is pathogenic. For example, C/EBPδ expression in Müller glia alone may cause the release
of a pathogenic factor resulting in PR cell death. To distinguish between the roles of C/EBPδ in
PRs vs. Müller cells, one could delete C/EBPδ in either PRs or Müller cells alone, as I described
above with reference to Stat-3. If the loss of C/EBPδ only in PRs of an IPD model resulted in a
slower rate of PR death, then C/EBPδ would be confirmed as a direct mediator of pathogenesis in
the mutant PR.
The downstream targets of C/EBPδ represent candidate genes involved in IPD PR death. A
microarray experiment to identify genes regulated by C/EBPδ might lead to the identification of
genes and pathways that are pathogenic to PRs.
The pathogenic role of C/EBPδ in PRs could have important therapeutic implications. If
IL-6 cytokines act directly on PRs, then pathogenic C/EBPδ expression may be an unavoidable
consequence of IL-6 treatment, which is protective overall. However, if Müller cells are the primary
mediators of PR protection as a result of IL-6 cytokine treatment, then IL-6 cytokines should be
screened for their ability to up-regulate C/EBPδ in PRs and not used as a potential therapy for
patients suffering from PR degeneration. Different IL-6 cytokines may have different capacities to
up-regulate C/EBPδ in PRs. PR cultures could be treated with each IL-6 cytokine and the degree of
C/EBPδ up-regulation could be assayed and compared. All other things being equal, the IL-6
cytokine that activates C/EBPδ in PRs the least may be the best candidate for human therapy. Some
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studies have indirectly addressed this question for Lif and Cntf. While Cntf treatment did not lead to
pStat-3 expression in PRs, Lif expression did, suggesting that Cntf might not be able to activate
C/EBPδ in PRs (Peterson et al., 2000; Ueki et al., 2008).
4.6 Reconciling the pathogenic and protective affects of IL-6 signalling
My finding in Chapter 3 that Lif loss-of-function decreased the rate of cell death in Rd1-/-
retinas was unexpected, since Lif is neuroprotective in both light-damaged and IPD models (LaVail
et al., 1998; Ueki et al., 2008). How can Lif be both protective and pathogenic in these different
contexts? While the lack of Lif during development had no effect on PR number in the work I
reported, it may have changed the responsiveness of PRs to the Rd1 mutation. Lif expression has
been shown to influence blood vessel formation in the retina (Ash et al., 2005; Kubota et al., 2008).
Transgenic mice expressing Lif exhibited a decrease in normal vasculature, resulting in retinal
ischemia and pathogenic neovascularization (Ash et al., 2005). In the Lif -/- murine retina, increased
microvessel density and a resistance to hyperoxic insults was documented (Kubota et al., 2008).
Since oxidative stress is observed in Rd1-/- retinas (Hackam et al., 2004b; Ahuja-Jensen et al., 2007;
Sharma and Rohrer, 2007), the loss of Lif in that model might also protect mutant PRs, accounting
for increased PR number in retinas of Rd1-/-;Lif-/- mice compared to Rd1-/-;Lif+/+ mice. Thus, an
alteration in the retinal vasculature may be a secondary effect of Lif loss-of-function, accounting for
the decreased rate of cell death. Mutant retinas treated with Lif, on the other hand, would not
exhibit altered vasculature and Lif could exert its protective affects.
4.7 Stat-3 may be involved in retinal regeneration in lower vertebrates
The regenerative ability of the adult mammalian retina is limited (Fischer and Reh, 2003). In
contrast, lower vertebrates can undergo retinal regeneration in response to injury (Gilbert, 2006).
For example, the retina of the teleost fish regenerates after surgical lesions to produce a functional
retina (Fischer and Reh, 2003). Similarly, the PR death elicited by light-damage is followed by
regeneration in zebrafish (Bernardos et al., 2007). Using a transgenic cell marking strategy, Müller
cells were observed to re-enter the cell cycle and produce progenitors that could differentiate into
rods and cones (Bernardos et al., 2007).
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To identify some of the molecular events occurring during the regeneration process,
microarray analysis was performed on retinal samples taken at several time-points after zebrafish
were exposed to damaging light (Kassen et al., 2007). Stat-3 was identified as significantly up-
regulated at all time-points, including during the periods of cell death, progenitor proliferation,
migration, and PR differentiation. Immunoblot analysis confirmed that pStat-3 was also up-
regulated; significantly, although pStat-3 was up-regulated greater than 3-fold at all time-points
examined, the peak of pStat-3 expression (~7 fold up-regulated) occurred at 68 hours post light
damage, a time-point characterized by progenitor proliferation and migration (Kassen et al., 2007).
Co-localization experiments demonstrated that the Stat-3 positive cells were Müller glia, a subset of
which also expressed PCNA, a marker of cell proliferation (Kassen et al., 2007). In this context, Stat-
3 may represent one of the first genes to be up-regulated during regeneration (Kassen et al., 2007).
The co-localization of PCDNA in a subset of Stat-3 labeled Müller glia may indicate that Müller glia
proliferation and differentiation occurs in an asynchronous fashion (Kassen et al., 2007).
Retinal regeneration has also been observed in the avian retina after cell death in the inner
retina was induced using N-methyl-D-aspartate (NMDA) (Fischer and Reh, 2003). Two days after
NMDA injection, a large number of mitotically active Müller glia were observed (Fischer and Reh,
2003). Fate mapping experiments suggested that Müller cells de-differentiate, undergo one round of
mitosis and then become one of three possible cell types: neurons, Müller glia, or undifferentiated
progenitor cells (Fischer and Reh, 2003). Subsequent studies showed that Müller cells are capable of
de-differentiation, cell division, and differentiation into ganglion cells (Fischer and Reh, 2003).
Similar experiments in rats completely lacking PRs after N-methyl-N-nitrosourea injection suggest
that some Müller derived cells expressed rhodopsin, possibly representing an attempt at regeneration
in the mammalian retina (Wan et al., 2008). Rodent Müller cells, grown under conditions normally
used for neural stem cell cultures, proliferate into sphere colonies and express genes normally found
in neuronal progenitor cells, confirming the ability of mammalian Müller glia to de-differentiate and
proliferate in vitro.
I initially hypothesized that the up-regulation of Stat-3 I observed in Rds+/- retinas was a
protective response. Given the aforementioned data, an alternative explanation is the increased Stat-
3 might represent a vestigial mammalian attempt to regenerate a damaged retina from Müller cells.
119
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