Diabetic retinopathy (DR) is the most common complication of diabetes and the leading
cause of visual impairment in the working-age population in the Western world (http://www.diabetesatlas.org/).
One of the earliest events observed in DR is the impairment of retinal perfusion,
caused by the constriction of retinal blood vessels, inducing biochemical and metabolic
alterations, perycites/endothelial degeneration, and retina ischemia (Wong et al.,
2016). Severe stages of DR include proliferative DR, caused by the abnormal growth
of new vessels, and diabetic macular oedema in the central part of the retina. DR
is strongly associated with a prolonged duration of diabetes, hypertension, and hyperglycaemia
but it can persist even after replacement of normal glycaemic status (Wong et al.,
2016). The fact that low-grade inflammation, particularly associated with the early
stages of DR, induces structural and molecular alterations which play critical roles
in the pathogenesis of DR is gaining attention in recent years (Mesquida et al., 2019).
Although DR is traditionally regarded as a microvascular disease, retinal neurodegeneration
is also involved. In particular, growing evidences indicate that the degeneration
of retinal neurons also occur before clinical signs of DR and the typical microvascular
alterations of DR may be a subsequent event (Wong et al., 2016; Simo et al., 2018).
However, at present, there are no decisive data confirming this scenario since the
timing of neuronal death and the rate of susceptibility of specific cell population
during DR is still controversial. In this article, we will highlight the latest findings
on the retinal ganglion cells (RGCs), the inner retinal heterogeneous neurons in which
the cell death related to DR is detected first (Simo et al., 2018), with the aim to
emphasize that the homeostasis of RGCs (or specific subpopulations of RGCs) could
be a main issue to mitigate hyperglycaemic damage in the retina.
DR affects RGCs: The importance of RGC death/survival in diabetes and the characterization
of RGCs suffering DR insult is a developing story, including therapies to inhibit
the neurodegeneration, although critical information on the cellular and molecular
events of DR dysfunction is yet needed. Recent evidences revealed that at early stage
of diabetes, deep morphological changes and electrophysiological defects take place,
mainly in the neurons of the inner retina (Sergeys et al., 2019). Indeed, diabetic
mice showed a reduction of neural retina affecting, in particular, the ganglion cell
complex, which includes nerve fiber layer, ganglion cell layer (GCL), and inner plexiform
layer, and the nerve fiber layer-GCL complex. The inner retinal thinning and loss-of-function
of inner retinal cells paralleled a significant decrease in RGC density and amacrine
cells (Sergeys et al., 2019). These time-dependent events can occur at the early stage
of DR and generally become more severe with the progression of diabetes.
By combining morphological, functional and genetic features, RGCs can be classified
into at least forty distinct types, each of which covers the mammalian retina to encode
reliably its part of the visual message (Christensen et al., 2019). The classification
into RGC type A–D (RGA, RGB, RGC, and RGD) subgroups is morphologically based on the
RGC soma sizes and dendritic characteristics. Functionally, RGCs can be defined ON-
and OFF-center RGCs as they respond best to increases and decreases in light intensity
delivered to their receptive field centers, while ON-OFF RGCs respond to both increases
and decreases in light intensity. In diabetic mice, early stage of hyperglycaemia
mainly altered the dendrites of RGCs arborizing in the ON sublamina of the inner plexiform
layer, with less significant changes in OFF sublamina (Cui et al., 2019). The subset
of RGCs ON-type RGA2 (ON-RGA2) displayed an appreciable reduction of the dendritic
field areas while OFF-RGA2 cells were unaltered. In addition, an increase in total
number of dendritic branches was detected both in ON- and OFF-RGA2 cells. Noteworthy,
ON and OFF signaling and interactions in the visual system are closely involved in
contrast detection. Electrophysiologically, only ON-RGA2 showed increased resting
membrane potentials and decreased membrane capacitance during diabetes although both
ON- and OFF-RGA2 displayed an enhanced excitability. This morphological and functional
remodeling could be explained as an attempt to balance the loss of presynaptic inhibitory
inputs, such as those generated by amacrine cells. In this respect, in non-pathological
conditions, RGA2 cells (equivalent to α-RGCs, the best recognized RGCs in mammalian
retina with the biggest size of soma and branching) exhibit a large receptive field
and show a short response latency/fast conducting axons, definitely playing an essential
role in visual processing. Likewise, the directionally selective RGCs have a key role
in the retina functions since they respond best to motion of a stimulus in a particular
direction. In diabetic mice, the ON-OFF direction-selective RGD2 cells, arborizing
both in ON and OFF sublaminae of inner plexiform layer, have a reduced branching in
ON-stratified dendrites (Cui et al., 2019). On the contrary, the ON-direction selective
cells RGC1, which are known to be resistant to pathological insults, were unaffected.
Glutamate excitotoxicity, through the overstimulation of mainly alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate
and N-methyl-D-aspartate (NMDA) receptors, which results in an uncontrolled intracellular
Ca2+ response in postsynaptic neurons and cell death, is a key initial process in
DR and RGCs loss (Simo et al., 2018). In the mammalian retina, alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate
and NMDA receptors are expressed by all RGCs. It has been suggested recently that
the early NMDA-induced insult may cause the reshape of RGCs dendrites by affecting
synaptic activity before RGCs apoptosis takes place (Christensen et al., 2019). Noteworthy,
a diverse susceptibility of different RGCs to NMDA excitotoxicity exists. Indeed,
NMDA overstimulation in mice altered mostly the J-RGCs (an OFF-RGC population expressing
the immunoglobulin superfamily recognition molecule named junctional adhesion molecule
B) while αRGCs being the most resistant type. Since the α-RGCs subtype RGA2 are particularly
damaged by diabetic insult (Cui et al., 2019), one may speculate that NMDA excitotoxicity
is not a key event mediating DR-induced RGA2 death. The fact that NMDA overstimulation
injured also BD- (ON-OFF RGCs sensitive to ventral motion) and W3-RGCs (the smallest
in size and the most numerous RGCs, functionally uncharacterized) indicates that RGCs
susceptibility is not correlated to the ON and OFF inputs, not even to their soma
and dendritic field size (Christensen et al., 2019). Among RGCs subtypes, melanopsin-expressing
RGCs (mRGCs) are intrinsically photosensitive neurons participating in “non-image
forming” visual processing in the retina, likely performing functions also beyond
circadian ones. In humans, mRGCs are characterized by large soma, located mainly in
the GCL and in smaller numbers in the inner nuclear layer, and wide dendritic arbor
(Obara et al., 2017). When compared with non-melanopsin RGCs, they seem to be more
resistant to glutamate toxicity occurring in several pathologies (Obara et al., 2017).
Although discrepancies have been reported in animal models, a relevant damage of mRGCs
was recently detected in patients with severe DR phase, showing not only symptoms
of visual impairment but also a compromised photo-entrainment of the circadian clock
(as for instance circadian misalignment, pupillary defects, and sleep disorder) (Obara
et al., 2017). In particular, the progression of DR correlates with a considerable
loss of RGCs, including an extensive reduction of mRGCs. The loss of mRGCs is more
pronounced in inner nuclear layer than GCL thus indicating a different damage among
nuclear strata. These data further indicated that hyperglycaemia effects can be detected
on RGCs as a key target and, of interest, these effects are cell subtype-dependent.
The possibility that mRGCs loss might occur prior to neurodegeneration of conventional
RGCs deserves to be investigated. The
Figure 1
summarizes DR-induced changes at dendritic field of distinct RGCs, although a systematic
picture on the fate and morphology of RGC subtypes during hyperglycaemia is still
to come.
Figure 1
Major changes induced by DR affecting the dendritic field of different RGCs.
RGC branching pattern and branching level in the five strata (S1–S2: OFF sublaminae;
S3–S5: ON sublaminae) of the inner plexiform layer (IPL) was depicted. The dendritic
arborization of melanopsin-expressing RGCs (mRGCs) located in the inner nuclear layer
(INL) was showed as well. DR: Diabetic retinopathy; GCL: ganglion cell layer; RGA:
RGC type A; RGC: retinal ganglion cell; RGD: RGC type D.
DR is a multifactorial pathology and items that play multiple roles in degenerative
cellular processes are taken into special consideration as possible targets for RGCs.
Apoptosis participates in the death of retinal neurons under different conditions,
including DR, but the catabolic pathway autophagy has been recognized also to be involved
(Amato et al., 2018). Using ex vivo mouse retinas treated with high glucose, we have
demonstrated recently that neurons which are committed to dye by apoptosis exhibit
an impairment of autophagic machinery, reflecting an altered equilibrium of apoptosis
and autophagy in the early phases of DR (Amato et al., 2018). In particular, in several
retinal cell populations of high glucose-treated explants, including RGCs, we observed
an increase of caspase-3 activity and a significant accumulation of autophagosomes.
The decrease of autophagic turnover being the consequence of mTOR (mammalian target
of rapamycin) activation, a master regulator of autophagy. To note, retina treatment
with octreotide, a well-known analogue of the neuropeptide somatostatin, preserved
hyperglycaemic neurons from apoptosis and, concomitantly, increased robustly the autophagic
activity inhibiting mTOR (Amato et al., 2018). These results suggest that somatostatin
is an endogenous pro-survival system in DR acting, at least in part, as an autophagy-modulating
factor. Different evidences support the neuroprotectant role of neuropeptides in retinal
diseases and re-balancing the dysfunctional cross-talk between apoptosis and autophagy
generated by injurious conditions is considered a key weapon in their arsenal (Cervia
et al., 2019). Accordingly, we have reported that apoptosis/autophagy equilibrium
can be a fruitful target for preserving RGCs and retinal cells in other pathological
conditions, as for instance oxygen-induced retinopathy (Cammalleri et al., 2017).
In line with this hypothesis, histone HIST1H1C, that play a central role in the apoptosis
machinery, was identified as a regulator of autophagy in DR (Wang et al., 2017). In
particular, histone HIST1H1C overexpressed in the retina of diabetic mice and promoted
aberrant autophagy maintaining the deacetylation status of H4K16. Histone HIST1H1C
was responsible also for retinal inflammation, gliosis, and loss of RGCs, similar
to the pathological changes identified in the early stage of DR. Its downregulation
rescued the diabetic-induced hallmarks, restoring physiological autophagy and attenuating
the death of RGCs (Wang et al., 2017). Confirming the coexistence of multiple processes
that differently contribute to RGCs apoptosis in DR, a role of Brg1/Notch axis was
observed recently (Zhang et al., 2019). Using in vitro RGCs administered with high
glucose, authors showed that Brg1 expression decreased. Brg1 (the central catalytic
subunit of chromatin-modifying enzymatic complexes) is a crucial modulator of gene
expression, since it participates in the control of chromatin dynamics essential for
physiological cellular processes. Brg1 upregulation enhanced the viability and reduced
the apoptosis of diabetic RGCs thus suggesting an additional target to preserve RGCs
during DR-induced damage. Of interest, it was observed that Brg1 protects RGCs by
increasing the activation of the transmembrane receptor Notch and its pro-survival
pathway, including the protein kinase Akt which is a well known regulator of autophagy
(Zhang et al., 2019).
Concluding remarks: Since increasing evidences indicate that the RGCs loss is an early
event in diabetes, a crucial point for therapies preventing DR is to avoid or mitigate
retinal neurodegeneration. The regulatory balance between death/dysfunction and life/function
in DR shifts as neuronal population changes. In this respect, further studies using
neuroprotective tools should broaden their scope beyond neuronal death to include
shape, dendritic arborization, intracellular pathways, and functions of RGCs. Indeed,
morphological and physiological changes in RGCs determine their ability to process
the photoreception and, in a certain extent, these alterations are responsible of
“image-forming” and non-image forming” visual defects occurring in DR. These are all
together primary aspects, especially for the development of diagnostic tools and therapeutics
approaches, which have the purpose to reduce retinal damage of diabetic patients.
This work was supported by grants from the Italian Ministry of Education, University
and Research: “PRIN2015” to DC and “Departments of Excellence-2018”Program (Dipartimenti
di Eccellenza) (Department for Innovation in Biological, Agro-food and Forest systems
- DIBAF - University of Tuscia, Viterbo, Italy) (Project “Landscape 4.0 - food, wellbeing
and environment”).