Neuroprotective pathways in the retina

A major cause of blindness in the Western world is degeneration of
photoreceptors as a result of point mutations in genes coding for
either phototransduction-related proteins or other proteins
important for retinal function. Despite the diversity of mutated
genes and proteins involved in this heterogeneous group of
progressive retinal dystrophies with homologous phenotypes, the
final event leading to blindness is apoptosis of photoreceptors.
This has led to intensive studies of the effects of neuroprotective
agents on the survival of photoreceptors in animal models of
retinitis pigmentosa. One such effective molecule discovered to
date to exert substantial rescue of retinal photoreceptors is glial
cell line-derived neurotrophic factor (GDNF). However, the
molecular mechanism of action underlying GDNF-mediated
neuroprotection remains unresolved. This dissertation and the
herein described studies were carried out with the goal of
elucidating neuroprotective mechanisms using the porcine retina as
a model. This species was selected due to its morphological and
anatomical similarities to human retina. In order to clarify
possible cellular mechanisms involved in neuroprotection, the
initial studies involved analysis of GDNF action in porcine retina.
It soon became evident that the GDNF-receptive cell in retina was
not the photoreceptor itself but rather retinal Mueller glial cells
(RMG), which are the major retinal glial cells. Thus, primary RMG
cell cultures prepared from porcine retina were established and
characterised to analyse this cell type without extraneous effects
from the retinal environment. Proteomic profiling revealed profound
changes in expression of RMG-specific marker proteins as an effect
of in vitro conditions. Thus, the in vitro experiments for studying
GDNF-induced signalling were performed with primary RMG cultures in
an early state (two weeks in vitro) in order to study cells
resembling the in vivo phenotype. GDNF was found to induce the ERK,
SAPK and PKB/AKT pathways, as well as upregulating basic fibroblast
growth factor (bFGF). Application of bFGF to primary porcine
photoreceptors in vitro promoted a concentration-dependent rescue.
Therefore a model of RMG-mediated indirect survival promoting
mechanism induced by GDNF could be proposed. The finding that RMG
are mediators of photoreceptor survival prompted further screenings
for RMG-specific, secreted molecules promoting photoreceptor
survival. A large-scale primary photoreceptor survival assay
(96well format) was developed, in which RMG-conditioned medium
(RMG-CM) was tested for survival activity. Conditioned medium was
observed as having specific photoreceptor survival-promoting
activity stemming from previously unidentified protein/s. Reducing
the complexity of RMG-CM by anionic chromatography revealed that
the activity does not bind to anionic resins. Mass spectrometric
identifications of the mono-Q flow-through identified 23 different
proteins from the active fraction, among them three potential new
candidates for neuroprotective activity in the context of
photoreceptor survival: connective tissue growth factor (CTGF),
insulin-like growth factor binding protein 5 (IGFBP5) and
insulin-like growth factor binding protein 7 (IGFBP7). Expression
cloning and re-testing of these candidates for their ability to
promote photoreceptor survival revealed that CTGF and IGFBP5 were
effective in protecting photoreceptors when applied in combination
with the RMG-conditioned media. Taken together, these results
indicate that such survival-promoting activity is multi-factorial.
RMG are likely to support photoreceptors by either cell to
cell-mediated paracrine signalling or by secreting factors into the
intercellular space between retina and retinal pigment epithelium,
which consists of a complex matrix of proteins and polysaccharides.
This matrix, designated as interphotoreceptor matrix (IPM),
directly borders three cell types: photoreceptors, RMG and the
retinal pigment epithelium and predisposes the IPM to function as
repository of neuroprotective molecules possibly secreted from
adjacent cells to protect and support photoreceptors. In order to
identify such novel neuroprotective substances, the composition of
IPM was investigated in this thesis by comparative proteomics. Over
140 different proteins were identified, the majority of which had
never been previously detected in the IPM. Among these, 13
candidates were found, which in other tissue systems have been
already reported to have a functional role in neuroprotection.