Microglia are not required for prion-induced retinal photoreceptor degeneration

Abstract

Degeneration of photoreceptors in the retina is a major cause of blindness in humans. Often retinal degeneration is due to inheritance of mutations in genes important in photoreceptor (PR) function, but can also be induced by other events including retinal trauma, microvascular disease, virus infection or prion infection. The onset of apoptosis and degeneration of PR neurons correlates with invasion of the PR cellular areas by microglia or monocytes, suggesting a causal role for these cells in pathogenesis of PR degenerative disease. To study the role of microglia in prion-induced retinal disease, we fed prion-infected mice a CSF-1 receptor blocking drug, PLX5622, to eliminate microglia in vivo, and the effects on retinal degeneration were analyzed over time. In mice not receiving drug, the main inflammatory cells invading the degenerating PR areas were microglia, not monocytes. Administration of PLX5622 was highly effective at ablating microglia in retina. However, lack of microglia during prion infection did not prevent degeneration of PR cells. Therefore, microglia were not required for the PR damage process during prion infection. Indeed, mice lacking microglia had slightly faster onset of PR damage. Similar results were seen in C57BL/10 mice and transgenic mice expressing GFP or RFP on microglia and monocytes, respectively. These results were supported by experiments using prion-infected Cx3cr1 knockout mice without PLX5622 treatment, where microglial expansion in retina was delayed, but PR degeneration was not. Contrary to predictions, microglia were not a causative factor in retinal damage by prion infection. Instead, newly generated PrPSc accumulated around the inner segment region of the PR cells and appeared to correlate with initiation of the pathogenic process in the absence of microglia.

Keywords: Cx3cr1 knockout; Degeneration; Gliosis; Macrophages; Microglia; Müller cells; PLX5622; Photoreceptors; Prion protein; Prions; Retina; Scrapie.

Fig. 1

Progressive retinal degeneration in tgGFP/RFP mice after intracerebral 79A scrapie inoculation. a H&E stain of retina from a mock-inoculated mouse at 162 dpi showing normal retinal structure. Yellow bar indicates thickness of outer nuclear layer (ONL), which contains nuclei of photoreceptor (PR) cells. b Retina from a 79A scrapie-infected mouse at 124 dpi showing thinning of ONL (yellow bar), alteration of the IS and OS regions and presence of an infiltrating mononuclear cell in the OS region (arrow). c Retina from a 79A scrapie-infected mouse at 153 dpi shows nearly complete degeneration of ONL (yellow bar) and loss of IS and OS regions. Thinning of inner nuclear layer (INL) and inner plexiform layer (IPL) is also apparent. GC = ganglion cell layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer with photoreceptor cell nuclei, IS = inner segment of photoreceptors, OS = outer segment of the photoreceptors, RPE = retinal pigment epithelium. Scale bar = 100um

Fig. 2

Histopathological changes in 79A scrapie-infected retinas. a TUNEL staining of mock-infected mouse shows no evidence of apoptosis. b Scrapie-infected tgGFP/RFP mouse retina with TUNEL-positive nuclei (arrow) in the ONL indicating active apoptosis at 124 dpi. c Scrapie-infected C57BL/10 retina showing TUNEL-positive nuclei in the ONL (arrow) at 144 dpi. d Anti-GFAP staining of mock-infected retina is restricted to perivascular astrocytes (arrow) in the GC layer. e & f In tgGFP/RFP and C57BL/10 scrapie-infected retinas, GFAP-positive (red) processes of activated Müller cells (astrocytes) extend from GC layer to outer limiting membrane (arrow) of the ONL. g Anti-Iba1 staining of mock-infected retina shows a few Iba1-positive microglia (arrows) in the IPL and OPL. h & i In both tgGFP/RFP and C57BL/10 scrapie-infected retinas, Iba1-positive microglia are now also in ONL, OS and IS regions (arrows). These cells have enlarged cell bodies and thick processes typical of activated microglia. Scale bar = 50 μm, each column contains images from one representative mouse

Fig. 3

Ablation of retinal microglia following PLX5622 treatment. Retinal flat mounts from uninfected tgGFP/RFP mice without drug treatment (a) and with PLX5622 treatment (b). a In untreated mice, GFP-expressing microglia span the entire retina and appear highly branched with fine delicate processes (inset). b After 7 days of PLX5622 treatment, microglia have disappeared from the entire retina. Scale bars = 200 μm for a and b, 20 μm for inset

Fig. 4

Scrapie-induced retinal degeneration in C57BL/10 mice in presence or absence of retinal Iba1-positive cells. On all graphs each dot denotes the mean value of counts from two sections of a single mouse, lines connect mean values at each timepoint, and lines end following euthanasia of final clinical mouse. PLX5622 treatment was initiated at 14dpi. a Number of infiltrating Iba1-positive cells in the photoreceptor layer increased in the no drug (ND) group from 124 to 162 dpi, whereas Iba1-positive cells were not observed in the PLX5622-treated group. b Apoptosis was detected by observation of TUNEL-positive cells in the outer nuclear layer (ONL) in both ND and PLX groups. Apoptosis was not dependent on presence of Iba1-positive cells. c ONL thickness (μm) decreased in both ND and PLX groups indicating that loss of photoreceptor nuclei in the ONL did not depend on Iba1-positive cells. ONL thinning in PLX group which lacked microglia appeared to be slightly faster than in the ND group. However, best-fit line slopes for points from ND and PLX groups between 118 and 164 dpi were not significantly different, p = 0.101

Fig. 5

Immunohistochemical staining of tgGFP/RFP retinas using anti-Iba1, anti-GFP and anti-RFP antibodies. a anti-Iba1 staining of uninfected retina shows absence of microglia in the ONL, IS, and OS, one Iba1-positive cell (red) is visible in the OPL (arrow). b In response to scrapie infection of retina, there is significant migration of Iba1-positive cells (arrow) to the ONL, IS and OS. c anti-GFP staining (brown), indicative of Cx3cr1 expression in microglial cells, is not present in the ONL, IS and OS of the uninfected retina. d In the infected retina, microglia expressing GFP (brown) are present at a similar density to Iba1-positive cells in (b). e anti-RFP staining (brown), specific for Ccr2-expressing monocytes is not visible in the uninfected retina. f A rare RFP-positive cell (arrow) is present in the IS of a scrapie-infected retina. Note the two unstained nuclei (arrowhead), which are likely Iba1/GFP positive cells. Scale bar is 25 μm

Fig. 6

Scrapie-induced retinal degeneration in tgGFP/RFP mice in presence or absence of retinal microglia. PLX5622 treatment was initiated at 90 dpi (red arrow). a Number of Iba1-positive cells in the photoreceptor layer increased in the no drug (ND) group, but remained low in the PLX5622-treated group. b The GFP-positive cells invading the OS and IS regions were also counted, as they indicated Cx3cr1 expression and a microglial phenotype. These were elevated at similar timepoints as were seen for Iba1. c Late in disease only rare cells were stained with anti-RFP antibody, which was a marker of Ccr2 expression and a monocyte-derived macrophage phenotype. d Apoptosis was detected by TUNEL staining in both ND and PLX groups in the ONL. e ONL of both ND and PLX groups showed marked thinning starting at 128 dpi. Comparison of ND vs PLX points around the maximum ONL thinning (129-134dpi, dotted boxes), was done by one tailed Mann-Whitney test, and p-values are shown on each panel

Fig. 7

Retinal flat mount study of tgGFP/RFP mice by confocal microscopy. a,b,c. Z-stacks of 40–50 optical 1 μm sections of retinal flat mounts from tgGFP/RFP mice which express GFP (green) in microglia and RFP (red) in monocytes. a Uninfected mouse shows scattered green microglia with long delicate processes. Cross-section below with XZ dimension shows most green cells are in the OPL-IPL areas. b At 159dpi infected mouse from ND group has a large increase in green microglia with larger cell bodies and stubby processes typical of activated microglia. Some green/red dual stained cells can also be seen. XZ cross-section shows many of these cells are now in the ONL/IS/OS region. c At 134 dpi an infected mouse from the PLX group shows reduced number of green microglia and a few green/red cells as well. In addition, a few round all red cells, probably monocytes can be noted (arrows). d-f Single optical sections from uninfected mouse show that microglia are mostly in the IPL and OPL layers. g-i Optical sections of 159 dpi ND mouse show that green microglia in the IPL and OPL layers have processes similar to those seen in uninfected mice, whereas in ONL,IS,OS region green microglia and green/red microglia appear activated and have stubby processes. Some cells have red material apparently internalized in phagolysosomes (inset). j-l. Optical sections of 134 dpi PLX mouse also have a few green microglia with processes in IPL and OPL layers. In ONL/IS/OS region round cells with red cytoplasm appear to be monocytes (arrow). All scale bars = 50 μm. Number of mice studied at each time-point for each group is indicated in the methods section

Fig. 8

Comparison of microglial migration to photoreceptor area and PR thinning in scrapie-infected mice with and without expression of Cx3cr1. a After 79A scrapie infection, a delay in the number of Iba1-positive cells migrating to the PR areas was seen in Cx3cr1 knockout mice vs tgGFP/RFP (Cx3cr1 heterozygous) mice. Levels became similar in both mouse strains after 145dpi. b No difference was noted in the timing of ONL thinning in Cx3cr1 knockout mice vs tgGFP/RFP mice, suggesting that the delay of microglia had no effect on timing of ONL degeneration. c and d Retinal flat mounts examined by confocal microscopy show green microglia in both tgGFP/RFP mice (c) and Cx3cr1KO mice (d). Both mice also had red/green cells thought to be green microglia which had phagocytosed rhodopsin and/or other outer segment material, which was seen as red autofluorescence [25, 27]. Cx3cr1 mice do not express any RFP, so the red in these mice could not be RFP. Scale bar = 25 μm

Fig. 9

PrPSc deposition in retinas at various times after 79A scrapie infection. All panels show tgGFP/RFP mouse retinal sections stained with D13 anti-PrP monoclonal antibody. a In a control uninfected mouse, PrP staining (brown) was seen in the IPL, INL and OPL regions. In addition, a faint tan stain could be seen in the IS region of the photoreceptor cells. All the staining in this field was likely to be normal host PrPC since the mouse was uninfected. Furthermore, these areas were not stained in PrP null mice. b At 104 dpi in aninfected mouse from the ND group additional new PrP staining in punctate granular accumulations was observed in the IS region (arrow). This material appeared to be disease-associated PrPSc. c-k. More detailed view of the IS region was seen at higher magnification. c Uninfected mouse with faint brown blush in IS region. d Eighty two dpi ND mouse with earliest appearance of clumped PrPSc staining in IS (arrow). e, f, g On subsequent days (104, 118, 131dpi) PrPSc appeared to accumulate progressively in the IS region (arrows), and small punctate deposits could also be seen in the ONL (yellow arrowheads). One mononuclear microglial cell was present in the OS layer (black arrowhead). h At 162dpi severe retinal degeneration was observed, and the ONL was reduced to a single layer of nuclei (yellow arrow). OS and IS regions were almost totally absent, and only faint PrPSc staining remained. i, j In PLX5622-treated mice at 104 and 118dpi, PrPSc staining was similar to staining in the ND mice. k At 134dpi severe retinal degeneration was seen. Two mononuclear cells with phagocytosed PrPSc were seen in the area of the former OS region (black arrowheads). These pictures are representative of the following mice: 4 uninfected ND, 16 infected ND and 10 infected PLX treated. Mice were examined at 9 timepoints between 67 and 163 dpi, and representative mice from timepoints showing typical early to middle retinal degeneration were shown in this figure. a, b scale bar = 100 μm, c-k scale bar = 25 μm