1. Microglial phagocytosis of living
photoreceptors contributes to inherited
retinal degeneration
Matthew K Zabel, PhD
Unit On Neuron-Glial Interactions in Retinal Disease
National Eye Institute, NIH
Disclosures: None
2. Introduction
• Retinitis pigmentosa- set of blinding hereditary diseases.
• Typically rod and then cone photoreceptor degeneration-
utility of gene therapy.
• The significant number and diversity of causative genes
underscores an advantage of finding broadly-shared
mechanisms.
• Clinical and laboratory findings indicate a role for non-cell
autonomous mechanisms such as inflammation.
– Microglial infiltration is found in both animal models and human
specimens.
– Suppression of pro-inflammatory and pro-oxidative properties
alleviates degeneration.
3. Microglia in RP
Model
•Rd10 mouse model.
•Well-characterized model
recapitulating features of
RP.
•Recessive mutation in rod
Pde6b.
• Infiltration of microglial
processes at peak of
photoreceptor apoptosis.
• TUNEL
• Transformation into
amoeboid morphology
• Development of multiple
phagosomes.
• Re-ramification
4. Phagocytic Role of Microglia in RP
• Expression of phagocytic
marker CD68 by infiltrating
microglia.
• Expression and secretion of
MFG-E8 by microglia and
labeling of ONL
photoreceptors.
• Expression of the “eat-me
signal” phosphatidylserine
on the outer leaflet of rods,
but not cones.
5. Microglial Phagocytosis of Live Rods
• Phagocytosed photoreceptors
are positive for Rhodopsin.
• Negative for Cone Arrestin.
• Peak phagocytosis coincides
with peak of microglial
infiltration and photoreceptor
apoptosis.
6. Microglial Phagocytosis of Live Rods
• Microglia phagocytosis is
primarily directed at non-
apoptotic rods
• Phagocytosed rods are:
• Negative for TUNEL
labeling
• Negative for early
markers of apoptosis:
activated caspase-3,
cleaved PARP
7. Alternate mouse models of RP and human RP
Mouse models
• Rd1
• Rd16
• RPGRIP
Human RP
• Autosomal Recessive
• Autosomal Dominant x2
• X-linked
8. • Live cell imaging of microglial
behavior in CX3CR1GFP/+, rd10
retinal explants
• Labeling with: GFP/Hoechst/PI
• Interaction of infiltrating
microglia with viable
photoreceptor nuclei
• Dynamic probing of
photoreceptor somata via
motile phagocytic cups
• Engulfment and phagocytosis of
photoreceptor soma and
translocation to microglial cell
body
• Migration of microglia within
ONL
Dynamic microglial phagocytosis of non-apoptotic rods
9. • Genetic model of
microglia depletion
• CX3CR1CreER x Rosa26-
flox-STOP-flox –DTA mice
in rd10 background
• Administration of
tamoxifen at P21 to
deplete microglia
• Sustained microglial
depletion delayed rod
degeneration
Contribution of Microglia to Photoreceptor
Degeneration: Depletion of Microglia
P28-29
P37-39
P50
10. Inhibition of Microglial Phagocytosis in rd10 retina
• Inhibition of phagocytic
vitronectin receptor with
cRGD peptide
• Ex vivo effects on rd10
retinal explants
• ↑ microglial ramification
• ↓ # of microglial
phagosomes
• In vivo effects on rd10
retina at P20
• ↓ # of infiltrating
microglia
• ↓ # of microglial
phagosomes
• ↑ photoreceptor rescue
11. Contribution of Infiltrating Microglia to
Photoreceptor Degeneration
• Microglia potentiate
degeneration by:
– Phagocytosis of stressed but
living rod photoreceptors in
non-cell autonomous
manner.
12. Conclusion
• Microglia play an active role in retinal degeneration
through:
1. Infiltration of the outer retina during photoreceptor
degeneration.
2. Expression of phagocytic machinery.
3. Dynamic interactions with viable photoreceptors.
• These microglial functions culminate in the phagocytosis of
stressed, but live rod photoreceptors, augmenting disease
progression.
• Targeting microglial phagocytosis will provide an opportunity to
slow progression of degeneration across the broad spectrum of
genetic etiologies.
13. Acknowledgements
UNGIRD
Wai T. Wong
Lian Zhao
Xu Wang
Wenxin Ma
Parth Shah
Biological Imaging Core
Robert Fariss
Functional Imaging Core
Hao-hua Qian
National Eye Institute, IRTA
funding
New York University
Christopher Parkhurst
Wen-Biao Gan
Foundation for Fighting
Blindness
Joe Hollyfield
Vera Bonilha
Retinal Cell Biology &
Degeneration
Tiansen Li
Editor's Notes
Mutations in many photoreceptor genes
Using the rd10 mouse model of retinitis pigmentosa to study the role of microglial involvement in disease progression
Infiltration via migration and radial process extension of microglia into the ONL coincides with photoreceptor apoptosis (thinning of the ONL) at P21.
Some infiltrating microglia transitioned into an amoeboid morphology and many microglia developed phagosomes.
REORDER FOR CLARITY AND SMOOTH
Because of the morphological changes that suggested a functional transition to phagocytic microglia, we stained similar sections for markers of phagocytic machinery and “eat-me” signals.
The phagolysosomal marker, CD68, showed increased expression in infiltrating microglia, especially around phagocytosed ONL nuclei at P22 (peak of degeneration and infiltration), which subsided by P30.
Similarly, the phagocytic bridging molecule, Milk Fat Globule Epidermal Growth Factor 8, localized to the ONL at P22 in a peri-somal distribution suggesting binding to targeted photoreceptors.
MFG-E8 binds to the “eat-me” signal PS, expressed by stressed but viable neurons (as shown in work by Guy Brown), which we find increased at P22 as well.
PS IHC localizes more to rods than cones
Because the PS “eat-me” signal labels rods almost exclusively, we labeled microglia w/ Iba1 and rods using rhodopsin or cones with cone arrestin and found that microglia selectively phagocytose rods over cones.
Microglia are thought to be solely “garbage-men” in disease processes (cleaning up apoptotic cells and debris) and this may be their role in RP as photoreceptors were dying at the same time as microglia infiltration. However, you will notice that microglia PHAGOCYTOSE TUNEL- PHOTORECEPTORS MORE PROMINENTLY THAN TUNEL+
This is also the case for early apoptotic markers caspase-3 and cleaved PARP.
To determine if this possible mechanism of non-cell autonomous death through microglial phagocytosis is evident in other forms of mouse and Human RP, we similarly stained such tissue.
EVIDENCE OF MICROGLIA UNDERGOING PHAGOCYTOSIS OF TUNEL - PHOTORECEPTORS IN THESE MODELS.
To catch these phagocytic microglia in action, we performed live imaging in retinal explants of GFP-labeled microglia in a rd10 background. We labeled the photoreceptor nuclei with Hoechst and non-viable photoreceptors with PI so that viable photoreceptors will be blue and not red.
We found that infiltrating microglia interact dynamically with viable photoreceptor somata via their processes that form transient phagocytic cups around the somata, contacting and releasing the photoreceptors repeatedly.
This motion is repeated multiple cycles and culminates in the overt engulfment of the photoreceptor into a phagosome and the translocation of the cell to the cell body of the microglia.
A single microglia can contact multiple photoreceptors by virtue of their process outreach but also by migrating within the ONL.
Also, the majority of contacted and phagocytosed photoreceptors were PI-negative rather than PI-positive, indicating this to be a mechanism primarily to clearing viable cells rather than dead cells.
To test whether microglia actually contribute directly to photoreceptor degeneration, we employed a genetic mouse model obtained from Dr Wenbiao Gan in which the CX3CR1 promoter in microglia drives the expression of inducible Cre activity. By crossing these mice into a flox-STOP-flox DTA in a rd10 background, we can induce the depletion of retinal microglia during rod degeneration to examine their role.
We initiated Cre activity at P21 by tamoxifen feeding and found that in a few days, substantial depletion of retinal microglia throughout the retina, and particularly in the outer nuclear layer was achieved to a substantial extent.
We maintained microglial depletion up to P28-29 and found that this resulted in a substantially greater preservation in the thickness of the ONL where photoreceptors are located compared to undepleted littermates. The extent of ONL preservation also negatively and significantly correlated with the total number of retinal microglia, indicating an association between microglial numbers and photoreceptor loss. The number of TUNEL+ ONL cells was also reduced with microglial depletion.
When we extended the period of depletion to longer time periods up to P37-39 and to P50, we continued to see significant morphological rescue effects, indicating that the delay in degeneration can be sustained over a longer time window.
We also assessed the ability of microglial depletion to effect functional rescue – we measured dark adapted and light adapted ERGs at P50, and found that microglial depleted retinas demonstrated significantly greater a- and b-wave amplitudes across a wide range of flash intensities.
These results indicate that infiltrating microglia do exert a significant contribution to the overall dynamics of photoreceptor degeneration in this RP model.
To test whether microglial phagocytosis was directly involved with photoreceptor cell death and ONL thinning, we blocked the phagocytic vitronectin receptor.
Ex vivo experiments show an increased microglial ramification and decreased number of total phagosomes and phagosomes per microglia after application of cRGD compared to cRAD.
We then inhibited microglial phagocytosis in vivo by injecting cRGD intravitreally into rd10 animals at P20; the contralateral eye of each animal was treated with the inactive analog, cRAD. When analyzed at P26, we found as we did ex vivo a decreased number of microglial phagosomes and with it, greater morphological rescue of photoreceptors.
When we performed ERG recordings and compared cRGD treated eyes with control cRAD treated eyes that there was a statistically increase in b-wave amplitudes, and to a lesser extent, a-wave amplitudes under dark- and light-adapted conditions.
These results indicated that phagocytosis per se conducted by microglia contributes to photoreceptor degeneration.
To summarize the contribution of microglia to retinal degeneration, although mutation-bearing rod photoreceptors can progress to apoptosis cell-autonomously, microglia can accelerate this process in a non-cell autonomous way.
Stressed but still living rods demonstrate exposure of PS, an “eat-me” signal and also likely the secretion of yet undefined “find me” signals.
Inner retinal microglia can detect this photoreceptor stress and migrate into the ONL where they express markers of activation and upregulate their phagocytic machinery.
Engaging with PS+ rods via dynamic process contacts, microglia can clear non-apoptotic rods via primary phagocytosis that can be inhibited by cRGD.
Through these influences, microglia can exert a potentiating effect on rod demise and accelerate photoreceptor degeneration.
